Treatment of extracellular matrix to reduce inflammation

ABSTRACT

Pharmaceutical compositions are provided. The compositions comprise a compound comprising the hyaluronan-containing structure A-(low molecular weight hyaluronan domain)-B. The compositions also comprise a pharmaceutically acceptable excipient. A is hydrogen, a substituent that does not comprise a binding site for tumor necrosis factor stimulated gene-6 (“TSG-6”) protein, a substituent that interferes with binding of TSG-6 protein immediately adjacent thereto, or chondroitin. B is hydroxyl, a substituent that does not comprise a binding site for TSG-6 protein, a substituent that interferes with binding of TSG-6 protein immediately adjacent thereto, or chondroitin. The composition is suitable for administration by injection, inhalation, topical rub, or ingestion. Also disclosed are methods and kits for treating a site of inflammation in an individual in need thereof and for decreasing heavy chain modification of pathological hyaluronan at a site of inflammation in an individual in need thereof based on administering the compounds or the compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/489,974, filed Jun. 6, 2012, which claims the benefit of U.S.Provisional Application No. 61/520,173, filed Jun. 6, 2011, the entiredisclosures of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to treatment of extracellularmatrix, and more particularly, to treatment of extracellular matrix toreduce inflammation.

BACKGROUND OF THE INVENTION

Inflammatory diseases, such as rheumatoid arthritis, affect millions ofpeople worldwide. In the United States, rheumatoid arthritis affectsmore than 1% of the population, at a cost of approximately $15,000 to$20,000 per patient per year. Currently there is no cure for rheumatoidarthritis, which eventually leads to permanent joint damage andfunctional disability. Conventional treatments involve medications usedto mediate the patient's immune system, globally affecting the immuneresponse, to reduce or slow inflammation. For example, corticosteroidsare used to reduce disease activity and joint inflammation acutely.However, their long-term use is inappropriate due to major side effectsof chronic use including diabetes, cataracts, weight gain, and adrenalsuppression. Corticosteroids are often used to control symptoms andflares of joint inflammation until disease-modifying anti-rheumaticdrugs (also termed DMARDs) reach their full effectiveness, which cantake up to 6 months.

Disease-modifying anti-rheumatic drugs can be divided into two generalcategories based on how they work: oral DMARDs and biological DMARDs.Oral DMARDs are taken by mouth. They interfere with the making orworking of immune cells that cause joint inflammation. Biological DMARDsare given by injection (infusion). They act in several different ways toaffect how immune cells work. DMARDs decrease joint inflammation anddamage. Because they work throughout the body to fight rheumatoidarthritis, they are also associated with serious side effects, includinglife-threatening infections, hematologic issues, kidney and liverdamage, severe immunosuppression and death, as well as long-term effectssuch as increased risk of cancer. Intense blood work is usually requiredto monitor the drug's effect. DMARDs are usually started within 3 monthsof diagnosis and are used to control the progression of rheumatoidarthritis and to try to prevent joint deterioration and disability.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin,ibuprofen, or naproxen are used to control pain and may help reduceinflammation. They do not control the disease or stop it fromprogressing. NSAIDs may be combined with DMARDs.

None of the drug-based therapies currently in use are successful inhalting the progression of inflammatory disease. Presently, the onlyoption at end stage disease is total joint replacement. Accordingly,novel therapies for the treatment of inflammatory diseases, slowing orstopping their advance, and reducing the incidence and need for jointreplacement surgeries, are desirable. Such therapies will preferablyhave a more localized action, and will not directly affect immune-systemfunction or pathways, and thus produce unwanted side effects.

During inflammation, serum exudates enter tissues, bathing themacromolecular hyaluronan (also termed hyaluronate, hyaluronic acid, orHA) found in most tissue extracellular matrices (ECM) with theserum-derived proteoglycan inter-α-inhibitor (IαI). Heavy chains (HC) ofIαI are transferred from IαI to HA by the enzyme tumor necrosis factorstimulated gene-6 (TSG-6), a protein expressed at sites of inflammation,causing a pathological transformation to the HA matrix. Thispathological transformation functions as a specific ligand forinflammatory cells. The formation of HC-HA complexes transform normallybenign HA matrix molecules into pathological HA that includes specificligands for inflammatory cell receptors contributing to inflammatorydisease processes through binding and activation of inflammatory cells.Strategies that would allow for the in vivo manipulation of HC transferboth to and from HA in inflamed tissues may provide a therapy fortreatment of a wide variety of inflammatory diseases.

BRIEF SUMMARY OF THE INVENTION

In one example aspect, a pharmaceutical composition is provided. Thecomposition comprises a compound comprising the hyaluronan-containingstructure:

The composition also comprises a pharmaceutically acceptable excipient.In accordance with the composition, A is selected from the groupconsisting of hydrogen, a substituent that does not comprise a bindingsite for tumor necrosis factor stimulated gene-6 (TSG-6) protein, asubstituent that interferes with binding of TSG-6 protein immediatelyadjacent thereto, and chondroitin. Also in accordance with thecomposition, B is selected from the group consisting of hydroxyl, asubstituent that does not comprise a binding site for TSG-6 protein, asubstituent that interferes with binding of TSG-6 protein immediatelyadjacent thereto, and chondroitin. Also in accordance with thecomposition, x=0 or 1, y=0 or 1, and n=3 to 70, with the proviso that ifn=3, then at least one of x and y=1. The composition is suitable foradministration by injection, inhalation, topical rub, or ingestion.

In another example aspect, a method of treating a site of inflammationin an individual in need thereof is provided. The method comprisesadministering to the site of the individual a therapeutically effectiveamount of a compound comprising the hyaluronan-containing structure:

In accordance with the method, A is selected from the group consistingof hydrogen, a substituent that does not comprise a binding site forTSG-6 protein, a substituent that interferes with binding of TSG-6protein immediately adjacent thereto, and chondroitin. Also inaccordance with the method, B is selected from the group consisting ofhydroxyl, a substituent that does not comprise a binding site for TSG-6protein, a substituent that interferes with binding of TSG-6 proteinimmediately adjacent thereto, and chondroitin. Also in accordance withthe method, x=0 or 1, y=0 or 1, and n=3 to 70, with the proviso that ifn=3, then at least one of x and y=1.

In another example aspect, a method of decreasing heavy chainmodification of pathological hyaluronan at a site of inflammation in anindividual in need thereof is provided. The method comprisesadministering to the site of the individual a therapeutically effectiveamount of a compound comprising the hyaluronan-containing structure:

In accordance with the method, A is selected from the group consistingof hydrogen, a substituent that does not comprise a binding site forTSG-6 protein, a substituent that interferes with binding of TSG-6protein immediately adjacent thereto, and chondroitin. Also inaccordance with the method, B is selected from the group consisting ofhydroxyl, a substituent that does not comprise a binding site for TSG-6protein, a substituent that interferes with binding of TSG-6 proteinimmediately adjacent thereto, and chondroitin. Also in accordance withthe method, x=0 or 1, y=0 or 1, and n=3 to 70, with the proviso that ifn=3, then at least one of x and y=1.

In another example aspect, a kit is provided. The kit comprises acompound comprising the hyaluronan-containing structure:

The kit also comprises a pharmaceutical excipient. The kit alsocomprises an instruction for use of the kit for treating a site ofinflammation in an individual in need thereof by administering to thesite a therapeutically effective amount of the compound. In accordancewith the kit, A is selected from the group consisting of hydrogen, asubstituent that does not comprise a binding site for TSG-6 protein, asubstituent that interferes with binding of TSG-6 protein immediatelyadjacent thereto, and chondroitin. Also in accordance with the kit, B isselected from the group consisting of hydroxyl, a substituent that doesnot comprise a binding site for TSG-6 protein, a substituent thatinterferes with binding of TSG-6 protein immediately adjacent thereto,and chondroitin. Also in accordance with the kit, x=0 or 1, y=0 or 1,and n=3 to 70, with the proviso that if n=3, then at least one of x andy=1.

In another example aspect, a kit is provided. The kit comprises acompound comprising the hyaluronan-containing structure:

The kit also comprises a pharmaceutical excipient. The kit alsocomprises an instruction for use of the kit for decreasing heavy chainmodification of pathological hyaluronan at a site of inflammation in anindividual in need thereof by administering to the site atherapeutically effective amount of the compound. In accordance with thekit, A is selected from the group consisting of hydrogen, a substituentthat does not comprise a binding site for TSG-6 protein, a substituentthat interferes with binding of TSG-6 protein immediately adjacentthereto, and chondroitin. Also in accordance with the kit, B is selectedfrom the group consisting of hydroxyl, a substituent that does notcomprise a binding site for TSG-6 protein, a substituent that interfereswith binding of TSG-6 protein immediately adjacent thereto, andchondroitin. Also in accordance with the kit, x=0 or 1, y=0 or 1, andn=3 to 70, with the proviso that if n=3, then at least one of x and y=1.

In another example aspect, a pharmaceutical composition is provided. Thecomposition comprises a compound comprising the structureA-(glcNAc*)_(x)-(glcA*-glcNAc*)_(n)-(glcA*)_(y)-B. The composition alsocomprises a pharmaceutically acceptable excipient. In accordance withthe composition, glcA* is D-glucuronic acid or substituted D-glucuronicacid, and glcNAc* is N-acetyl-D-glucosamine or substitutedN-acetyl-D-glucosamine, with the proviso that(glcNAc*)_(x)-(glcA*-glcNAc*)_(n)-(glcA*)_(y) includes at least onebinding site for TSG-6 protein. Also in accordance with the composition,A is selected from the group consisting of hydrogen, a substituent thatdoes not comprise a binding site for TSG-6 protein, a substituent thatinterferes with binding of TSG-6 protein immediately adjacent thereto,and chondroitin. Also in accordance with the composition, B is selectedfrom the group consisting of hydroxyl, a substituent that does notcomprise a binding site for TSG-6 protein, a substituent that interfereswith binding of TSG-6 protein immediately adjacent thereto, andchondroitin. Also in accordance with the composition, x=0 or 1, y=0 or1, and n=3 to 70, with the proviso that if n=3, then at least one of xand y=1. The composition is suitable for administration by injection,inhalation, topical rub, or ingestion.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other aspects of the present invention will becomeapparent to those skilled in the art to which the present inventionrelates upon reading the following description with reference to theaccompanying drawings, in which:

FIG. 1 is a schematic illustration of irreversible heavy chain transferto LMW HA. (A) Inter-α-Inhibitor (IαI) has two HCs attached to theproteoglycan bikunin by covalent bonds with chondroitin sulfate (CS).TSG-6 removes a HC from IαI to form a HC-TSG-6 intermediate andtransfers the HC onto the much larger HMW HA. (B) When “naked” HMW HA ismixed with HMW HC-HA, TSG-6 shuffles HCs between the two HA strandsuntil the HCs are evenly distributed between the two strands. (C) Whennaked LMW HA is mixed with HMW HC-HA, TSG-6 transfers HCs onto LMW HAbut it is unable to transfer them back to HMW HA.

FIG. 2 is a schematic illustration of irreversible transfer of heavychains (HC) from high molecular weight hyaluronan (HMW HA) to lowmolecular weight (LMW) HA via TSG-6. (A) HA is synthesized as a “naked”carbohydrate (i.e. lacking covalent binding partners) at the surface ofcells. During inflammation, TSG-6 covalently transfers HC fromserum-derived inter-α-inhibitor onto HA. HC covalently bound to HA canbe shuttled between HMW HA chains by TSG-6 until they reach a reversibleequilibrium. (B) LMW HA, such as HA that is 10 monosaccharides long(HA10) are also acceptors for HC transfer via TSG-6, but unlike HMWHC-HA, LMW HC-HA cannot serve as a donor for HC transfer. Thus, thetransfers of HCs to LMW HA is irreversible.

FIG. 3 shows schematically the irreversible effect of LMW HA (e.g.composed of 8 to less than about 160 monosaccharides) on TSG-6-catalyzedHC transfer to HA. LMW HA (e.g. HA composed of 8 monosaccharides asillustrated) is able to bind to TSG-6 and accept transfer of HC fromTSG-6. However, once released from the TSG-6 enzyme, LMW HC-HA is itselfunable to bind to TSG-6. Thus LMW HA can serve as a HC acceptor, but notHC donor.

FIG. 4 shows schematically the lack of TSG-6 catalyzed HC transfer ontoHA6 or less. HA composed of 6 or fewer monosaccharides does not acceptHCs via a TSG-6-mediated reaction. This is believed to be because thesmall HA molecule is either: (A) too small to bind to the TSG-6 HAbinding site; or (B) upon binding is too small to accept transfer of HCfrom TSG-6.

FIG. 5 shows schematically the reversible effect of HA160 or more (alsotermed HA160+) on TSG-6 catalyzed HC transfer to HA. HA composed of ˜160monosaccharides or more is able to bind to TSG-6 and accept transfer ofHC from TSG-6. Once released from the TSG-6 enzyme the HC-HA160+ is ableto bind to TSG-6 and transfer its HC back to TSG-6. The HA160+ withoutHC is then released by the enzyme. Thus HA composed of 160monosaccharides or more can serve as both a HC acceptor and donor.

FIG. 6A-D shows results for kinetics of transfer of heavy chains (HCs),in which Western blots show the molecular weight gel shift of HCstransferred from inter-α-inhibitor (IαI) to hyaluronan (HA) and swappingbetween HA molecules of various sizes. Initial transfer of HC from IαIto (A) HA8, (B) HA16K, (C) HA50K, or (D) HA1000K, followed byintroduction, at various time points, of (A) HA16K or (B-D) HA8.

FIG. 7 illustrates results of a preliminary range study for theirreversible transfer of heavy chains to hyaluronan oligosaccharides, inwhich Western blots show the molecular weight gel shift of heavy chains(HCs) transferred from inter-α-inhibitor (IαI) and pre-IαI to hyaluronan(HA) and swapping between HA molecules to HA oligosaccharides. (A)Western blot with mouse serum. (B) Western blot with human serum.

FIG. 8 shows results of irreversible transfer of heavy chains from HMWHA to LMW HA via TSG-6. (A) Western blot showing the molecular weightgel shift of HCs transferred from inter-α-inhibitor (IαI), pre-IαI, orHMW HA to LMW HA. (B) Quantification of the HC band from the Westernblot, in which error bars represent standard deviation (n=3).

FIG. 9 shows modification of tyramine-substituted hyaluronan (T-HA) withheavy chains (HC) from inter-α-inhibitor (IαI) via TSG-6, by Westernblot. (A) Modification of HA, followed by hyaluronidase treatment. (B)Modification of T-HA, followed by hyaluronidase treatment. (C)Modification of HA, without subsequent hyaluronidase treatment. (D)Modification of T-HA, without subsequent hyaluronidase treatment.

FIG. 10 shows an analysis of heavy chain complex in synovial fluid fromrheumatoid arthritis patients, by Western blot.

FIG. 11 shows results of irreversible transfer of heavy chains from HMWHA to LMW HA in the synovial fluid of rheumatoid arthritis patients, bya Western blot of SF probed with an antibody that binds only heavychains (HCs).

FIG. 12 shows evidence for LMW HA in synovial fluid and serum fromrheumatoid arthritis patients. (A) Synovial fluid (SF) from fourpatients (a-d) was treated +/− with hyaluronidase and the amount ofreleased HCs analyzed by Western blot. (B) Serum from these samepatients was treated α/− with TSG-6 (panel B).

FIG. 13 shows results for irreversible transfer of human serum heavychains to hyaluronan oligosaccharides, by Western blot.

FIG. 14 shows results of irreversible transfer of heavy chains frommouse synovial fluid hyaluronan to hyaluronan oligosaccharides, byWestern blot.

FIG. 15 shows results of a size-range study of irreversible heavy-chaintransfer, by Western blot.

FIG. 16 shows results of another size-range study of irreversibleheavy-chain transfer, by Western blot.

FIG. 17 shows results of another size-range study of irreversibleheavy-chain transfer, by Western blot.

FIG. 18 shows results of kinetics of transfer of serum heavy chains 1and 2 to hyaluronan oligosaccharides, by Western blot. (A) Human HC1.(B) Human HC2. (C) Mouse HC2. Molecular weight (MW) markers are 75, 100,150, 250 kDa.

FIG. 19 shows results of heavy chain-hyaluronan complex promotinginflammatory cell adhesion. The HA-HC complex (B), in contrast to HAalone (A), promoted leukocyte adhesion. This adhesion was disrupted bydigestion of the HC-HA complex with hyaluronidase (C). Similar resultswere observed from HC-HA isolated from the synovial fluid of rheumatoidarthritis patients (17).

FIG. 20 shows analysis of heavy chain-hyaluronan complex in mouseproteoglycan-induced arthritis synovial fluid (PGIA SF), by Westernblot. Molecular weight (MW) markers are 75, 100, 150, 250 kDa.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that in the presence of TSG-6, HC can be irreversiblytransferred from high molecular weight (HMW) HA to low molecular weight(LMW) HA thereby returning the pathological HA in the ECM to itsoriginal state (FIGS. 1 and 2). This is significant because pathologicalHA in the ECM of living tissues is HMW HA, which is typically in amolecular-weight range of, e.g., 1,000-5,000 kDa, but which can begreater than 10,000 kDa. For HMW HA, the TSG-6 mediated transfer of HCbetween HA molecules is a reversible process, resulting in the evendistribution of HC along the lengths of all HMW HA chains present (FIGS.1B and 2A). However, LMW HA (e.g. 8 to less than about 160monosaccharides long) while also acceptors for HC transfer via TSG-6,cannot serve as donors for HC-transfer (FIGS. 1C and 2B). Irreversibleswapping of HCs from HMW HA to LMW HA can potentially be exploited toremove HCs from pathological HC-HA matrices found in RA and otherinflammatory diseases. The resulting LMW HA, modified with HC, isexpected to be readily cleared from the joint space. This will remove HCfrom the joint matrix, and thus the leukocytes will not be retained.Moreover, HA molecules are neither known nor predicted to be toxic,antigenic, or allergenic. This provides a paradigm for quantitativelyremoving pathologic HC ligands from HA of inflamed tissues, potentiallyalleviating or eliminating inflammation, and providing patient relief byremoving inflammatory-cell recruitment sites in the ECM of the affectedjoint or tissue. Administration of formulations of LMW HA in an affectedjoint or other tissue with exogenously supplied recombinant TSG-6 orendogenous TSG-6 would constitute a novel class of therapeutics fortreatment of inflammatory conditions such as rheumatoid arthritis,psoriatic arthritis, psoriasis, inflammatory bowel disease, asthma,etc., and could potentially be used as a prophylactic to prevent initialonset of inflammatory disease.

For purposes of this disclosure, LMW HA refers to HA molecules having 8to less than about 160 monosaccharide units, or a molecular weight inthe range of about 1.5 kDa to less than about 30 kDa. We have shownparticularly that LMW HA having 8 to 87 monosaccharides is effective inthe disclosed methods. Without wishing to be bound by theory, it isbelieved that LMW HA is able to bind to TSG-6 and accept transfer of HCfrom TSG-6, but that once released from the TSG-6 enzyme, LMW HC-HA isitself unable to bind to TSG-6, and thus that LMW HA can serve as a HCacceptor, but not HC donor (FIG. 3).

In contrast, HA molecules having 6 monosaccharides have been found to beunable to receive HCs via TSG-6-mediated transfer from tissue HA.Without wishing to be bound by theory, it is believed that HA moleculesof 6 monosaccharides or smaller do not provide sufficient length foreither binding to the TSG-6 enzyme, or if bound to the enzyme, for theenzyme to transfer a HC to the bound HA molecule (FIG. 4).

Of note, because HA is a molecule composed of repeat disaccharide units(D-glucuronic acid and N-acetyl-D-glucosamine linked via glycosidicbonds), it will not naturally occur in odd-numbered monosaccharideunits. However, it may be possible to prepare a synthetic HA-analoghaving the same structure as HA but with only 7 monosaccharide units;i.e. excluding either a terminal D-glucuronic acid orN-acetyl-D-glucosamine residue. (It will be naturally recognized topersons of ordinary skill in the art that references to hyaluronan,hyaluronic acid, hyaluronate, or HA are understood to includeconventional HA as well as such HA analogs having an odd number ofmonosaccharide units, for example due to the cleavage of a terminalmonosaccharide from the HA chain). It is believed that a7-monosaccharide HA analog may also serve as LMW HA in the disclosedmethods.

At the other end of the spectrum, we have determined that HA moleculeshaving about 160 or more monosaccharides do not function like LMW HA inthe disclosed methods because although they do accept HC transfer via aTSG-6-mediated reaction, they do so reversibly (FIG. 5).

Again without wishing to be bound by theory, it is believed that thereis a range of HA lengths wherein the chain is long enough to initiallybind TSG-6 so as to mediate the attachment of a HC, but not long enoughfor TSG-6 to bind again once the HC has been attached, so as to mediatethe removal of the previously transferred HC. This range will constitutethe range of permissible HA lengths (molecular weights) in the disclosedmethods. As noted above, currently, the range of 8-87 monosaccharides isknown to work as LMW HA molecules in the disclosed methods; that is, toirreversibly accept HCs transferred from tissue HA (which is HMW HA) viaa TSG-6-mediated reaction. In preferred embodiments, the LMW HA willrange from 8-25 monosaccharides in length, more preferably 8-20, 8-15 or8-10 monosaccharides. However, LMW HA with monosaccharide lengthsranging up to 130 monosaccharide units (i.e. up to 25 kDa), andapproaching less than about 160 monosaccharide units (i.e. less thanabout ˜30 kDa), may be effective in the disclosed methods as LMW HAmolecules.

For HA molecules in the range of 88 to less than about 160monosaccharides, there may be a range where the molecules function asLMW HA in the disclosed methods depending on reaction kinetics and theprecise location along the HA chain where the HC(s) is/are transferred.For example, based on the aforementioned TSG-6 transfer-site theory, ifa HC is transferred to a 130 monosaccharide HA near a terminus, or ifmultiple HCs are transferred in close proximity to one another (e.g.within 6-8 monosaccharide units of length), there may be insufficientroom for TSG-6 to re-bind to the HA adjacent the attached HC, resultingin a permanent transfer such that the HA molecule acts as LMW HA for thepresent methods. On the other hand, such chain lengths of 130monosaccharides may provide sufficient binding space for TSG-6 adjacenta prior-attached HC moiety so that the attachment is reversible. In thelatter instance, the HA molecule will not serve as LMW HA in the presentmethods. From the above, however, it will be appreciated that there willbe a range of monosaccharide length (molecular weight) for HA moleculeswhere they may transiently (i.e. under certain circumstances) behave asLMW HA so as to irreversibly accept HCs transferred from tissue HAaccording to the disclosed methods.

In practice, particularly in providing a therapy for treatment ofinflammation, it will generally be desirable to select HA moleculeswhose length/molecular weight is sufficiently low so that it behavesconsistently as LMW HA to irreversibly accept HCs as herein described.In addition to being easier to make and easier for the body to clearfrom an inflammatory site via natural processes, such LMW HA moleculeswill consistently irreversibly accept HCs, providing more efficienttherapies based on known concentrations of LMW HA delivered. This is thereason that HA molecules of 8-25 monosaccharides in length arepreferred. However, there may be instances where larger HA moleculeshaving less than about 160 monosaccharides (i.e. <˜30 kDa) could be usedas the LMW HA in the disclosed methods. It is believed that HA moleculeshaving about 160 monosaccharides or greater will consistently exhibitbulk reversible HC-transfer, making them unsuitable for use as LMW HA inthe disclosed methods.

Based on the foregoing, the molecular weight of a LMW HA will be lessthan about 30 kDa. A HMW HA is one whose molecular weight is about 30kDa or greater.

There is a kinetic component to the meaning of the term irreversible, asused herein. As can be seen in FIG. 6A, when HCs are transferred byTSG-6 from IαI to HA8 (HA that is 8 monosaccharides long) for differenttime periods before introducing HA16K (HA that is ˜16 kDa in size), asmall portion of HCs are transferred from the HC-HA8 to HA16K, which canbe seen as a very faint band in lane 4, migrating where HC-HA16 kDa ismarked on the right of the gel, that diminishes in intensity as timeprogresses. This suggests the presence of a small portion of HCs onHC-HA8 that are located on reversible sites within HA8. An alternativeexplanation is that HC transfer from IαI to HA8 was not complete at 4hrs and that the HC-HA16 kDa band in lane 4, and thereafter, came fromIαI and not HC-HA8. Regardless, the small proportion of HCs on HC-HA16kDa decreases with incubation time, emphasizing the irreversible qualityof HA8. As can be seen in FIG. 6B, when HCs are transferred by TSG-6from IαI to HA16K for different times before introducing HA8, a smallportion, but greater than that transferred from HA8 in FIG. 6A, of HCsare transferred from the HC-HA16K to the HA8 at the early time points,decreasing with time before addition of the HA8. Without wishing to bebound to theory, our interpretation is that because the HA16K is largerthan the HA8, there is initially a greater proportion of reversible HCson the HA16K than for the HA8, and it takes longer for these reversibleHCs to be swapped by TSG-6 to irreversible sites on the HA16K. However,note that for both HA8 and HA16K, which are considered LMW HA domainscapable of being HC acceptors but not donors, the majority of HCtransfer from IαI is initially irreversible, with the degree ofirreversibility increasing with time. As can be seen in FIG. 6C, whenHCs are transferred by TSG-6 from IαI to HA50K (HA that has a molecularweight of 30.6 kDa as determined by multi-angle laser light scatteringsize exclusion chromatography, also termed MALLS-SEC) for differenttimes before introducing HA8, the majority of the HCs are transferableto the HA8 as HA50K is considered HMW HA that is both a HC acceptor anddonor. A similar result is seen in FIG. 6D for HA1000K (HA that is ˜1000kDa in size). Note, for HA50K and HA1000K, while they are considered HMWHA, and thus not irreversible HC acceptors, but rather HC acceptors anddonors as used herein, not all of the HCs have been swapped from HA50Kand HA1000K to HA8 when the HCs were first transferred to HA50K andHA1000K for 24 hours before the addition of HA8, suggesting that evenHMW HAs such as HA50K and HA1000K may contain small amounts of HCsattached irreversibly. Alternatively, a longer incubation period than 72hrs may be required to remove all of the HCs from HC-HA50K andHC-HA1000K. Thus there is a temporal and size component toirreversibility as used herein.

Considering example aspects of the compositions, methods, and kitsdisclosed herein in more detail, in one example aspect a pharmaceuticalcomposition is provided. The composition comprises a compound comprisingthe hyaluronan-containing structure:

In accordance with the composition, A is selected from the groupconsisting of hydrogen (i.e. H), a substituent that does not comprise abinding site for TSG-6 protein, a substituent that interferes withbinding of TSG-6 protein immediately adjacent thereto, and chondroitin.

The substituent that does not comprise a binding site for TSG-6 proteincan be, for example, a monosaccharide, oligosaccharide, polysaccharide,alkyl group, or functional group, or a modified or derivatized versionthereof, that is not recognized as a binding site by TSG-6 protein andthus to which TSG-6 protein cannot bind. Based on the disclosure herein,one of ordinary skill would be able to identify and confirm any of awide range of substituents for use in this regard without undueexperimentation or effort.

The substituent that interferes with binding of TSG-6 proteinimmediately adjacent thereto can be, for example, a bulky adduct, amonosaccharide substituted with a bulky adduct, or a polysaccharidesubstituted with a bulky adduct, the bulky adduct being positionedimmediately adjacent the remainder of the structure. The bulky adductcan be, for example, a tyramine group or a hydroxyphenyl group. Themonosaccharide substituted with a bulky adduct can be, for example, aD-glucuronic acid residue, substituted with a tyramine group or ahydroxyphenyl group. The polysaccharide substituted with a bulky adductcan be, for example, HMW hyaluronan substituted by one or more tyramineor hydroxyphenyl groups, at least one of the tyramine or hydroxyphenylgroups being positioned immediately adjacent the remainder of thestructure.

By the substituent interfering with binding of TSG-6 protein, it ismeant that the substituent prevents TSG-6 protein from binding the partof the structure immediately adjacent to the substituent, at least tothe extent of preventing TSG-6 from removing an IαI heavy chain that hasbeen attached to that part of the structure.

Chondroitin is a glycosaminoglycan that includes a disaccharide repeatof N-acetylgalactosamine and glucuronic acid. The disaccharide ofchondroitin is thus identical to that of HA except for the conformationof one hydroxyl group. Chondroitin can be bound by TSG-6, withsubsequent heavy chain transfer, though less effectively than for HA.

Also in accordance with the composition, B is selected from the groupconsisting of hydroxyl (i.e. OH), a substituent that does not comprise abinding site for TSG-6 protein, a substituent that interferes withbinding of TSG-6 protein immediately adjacent thereto, and chondroitin.As one of ordinary skill will recognize from the structure as drawn, incontrast to A, which is linked to the remainder of the structure via thenon-reducing end of a saccharide, B is linked to the remainder of thestructure via the reducing end of a saccharide. Taking this differenceinto account, the substituent that does not comprise a binding site forTSG-6 protein and the substituent that interferes with binding of TSG-6protein immediately adjacent thereto for B otherwise can be, forexample, as described above for A.

Also in accordance with the composition, x=0 or 1, y=0 or 1, and n=3 to70, with the proviso that if n=3, then at least one of x and y=1.

As one of ordinary skill will appreciate from the foregoing, thehyaluronan-containing structure comprises a LMW HA domain between A andB. Specifically, this structure comprises the disaccharide repeatingunit of hyaluronan, i.e. a disaccharide with alternating β (1-3)glucuronidic acid and β (1-4) glucosaminidic bonds, in this case with adegree of polymerization of 3 to 70. The disaccharide is flanked, at itsnonreducing end, by A and optionally an N-acetyl-D-glucosamine residuetherebetween. The disaccharide is also flanked, at its reducing end, byB and optionally a D-glucuronic acid residue therebetween. As will beappreciated, if the degree of polymerization of the disacchariderepeating unit is 3, then at least one of the flankingN-acetyl-D-glucosamine residue or D-glucuronic acid residue must bepresent. Accordingly, the structure includes at least seven consecutivemonosaccharides of hyaluronan between A and B. As will also beappreciated, if the structure is not terminated by hydrogen at itsnonreducing end, it will be terminated there by the substituent thatdoes not comprise a binding site for TSG-6 protein, the substituent thatinterferes with binding of TSG-6 protein immediately adjacent thereto,or chondroitin. Likewise, if the structure is not terminated by hydroxylat its reducing end, it will be terminated there by the substituent thatdoes not comprise a binding site for TSG-6 protein, the substituent thatinterferes with binding of TSG-6 protein immediately adjacent thereto,or chondroitin. Accordingly, the structure does not include more than142 consecutive monosaccharides of hyaluronan between A and B, nor canthe part of the structure between A and B be recognized by TSG-6 as ifthe part were HMW HA.

The composition also comprises a pharmaceutically acceptable excipient.A pharmaceutically acceptable excipient refers to an excipient thatcauses no significant adverse toxicological effects to the patient.Suitable pharmaceutical excipients include, for example, water forinjection, carbohydrates, inorganic salts, antimicrobial agents,antioxidants, surfactants, buffers, acids, bases, divalent cations (e.g.Mg⁺² or Mn⁺²), and combinations thereof.

The composition is suitable for administration by injection, inhalation,topical rub, or ingestion. As used herein, suitable for administrationby injection, inhalation, topical rub, or ingestion means that thecomposition would be considered acceptable for therapeutic application,e.g. for a human, a dog, a cat, or a horse, by injection, inhalation,topical rub, or ingestion. In this regard, the pharmaceuticalcomposition typically would need to be sterile, substantiallypyrogen-free, and have no medically unacceptable effects. For example,the composition should not produce a medically unacceptableimmunological reaction when injected into a human subject. Medicallyunacceptable effects can be determined by one of ordinary skill in thefield of medicine. The composition can be made suitable foradministration by injection, inhalation, topical rub, or ingestion byroutine methods that are known in the art, such as purification of thecompound, the pharmaceutically acceptable excipient, and othercomponents prior to addition to the composition, and formulating,storing, and testing the composition by standard methods, and thus canbe obtained without undue experimentation or effort.

Considering the compound in more detail, the compound can be oneproduced by a non-biological process. For example, as discussed indetail below, LMW HA can be made enzymatically by published and patentedprocesses, based on extension of short HA chains (or primers) into muchlonger HA chains by recombinant Escherichia coli-derived HA synthase ofPasteurella multocida (PmHAS). These approaches offer benefits such asprecise control over the degree of polymerization of the LMW HAmolecules and higher purity of final product in comparison to biologicalprocesses for production and purification of HA.

The compound can be, for example, one in which A is hydrogen and B ishydroxyl, thus corresponding to a LMW HA without any non-saccharidesubstituents.

Also for example, the compound can be one in which A is the substituentthat does not comprise a binding site for TSG-6 protein, A being asaccharide, and B is the substituent that does not comprise a bindingsite for TSG-6 protein, B being a saccharide. Such a compound mayinclude as A and B, for example, saccharides other than those thatnaturally occur in HA and that do not otherwise include a binding sitefor TSG-6 protein.

Also for example, the compound can be one in which A is the substituentthat interferes with binding of TSG-6 protein immediately adjacentthereto, A being hyaluronan modified with tyramine, and B is thesubstituent that interferes with binding of TSG-6 protein immediatelyadjacent thereto, B being hyaluronan modified with tyramine. Forexample, the compound can be hyaluronan with a tyramine substitution ofabout 1 tyramine adduct about every 20 to 90 monosaccharides, or aboutevery 30 to 50 monosaccharides, or about every 40 monosaccharides. Asone of ordinary skill will appreciate based on the disclosure herein,TSG-6 protein will interact with such hyaluronan modified with tyramineby adding HC chains irreversibly, as though the parts of the hyaluronanmolecules between the tyramine substitutions were LMW HA, i.e.attachment of HC to these parts by TSG-6 will be irreversible.

Also for example, the compound can be, for example, one in which A ischondroitin and B is chondroitin, thus corresponding to a chimera ofchondroitin-LMW HA-chondroitin. In such a chimera, the chondroitin mayserve the purpose of interrupting, or setting off, the LMW HA, ensuringthat it is recognized as a LMW HA domain by TSG-6.

The composition can be one wherein, e.g. n=3 to 65 (corresponding toapproximately 7 to 132 monosaccharide units), n=3 to 45 (correspondingto approximately 7 to 92 monosaccharide units), or n=3 to 12(corresponding to approximately 7 to 26 monosaccharide units). Thecomposition can also be one wherein n=41 to 45 (corresponding toapproximately 82 to 92 monosaccharide units), or n=43 to 44(corresponding to approximately 86 to 90 monosaccharide units).

The composition can be, for example, one wherein A is hydrogen; B ishydroxyl; x, n, and y are selected from the group consisting of: (i)x=0, n=4, and y=0; (ii) x=0, n=5, and y=0, (iii) x=0, n=6, and y=0; (iv)x=0, n=7, and y=0; (v) x=0, n=9, and y=0; (vi) x=1, n=10, and y=0; and(vii) x=0, n=10, and y=1. The composition can also be, for example, onewherein A is hydrogen; B is hydroxyl; x, n, and y are selected from thegroup consisting of: (i) x=0, n=43, and y=0; (ii) x=1, n=43, and y=0,(iii) x=0, n=43, and y=1; (iv) x=1, n=43, and y=1; and (v) x=0, n=44,and y=0.

The composition can include the compound in a particular range ofmolecular weights, i.e. the composition can include more than onestructural embodiment of the compound. For example, the composition canhave a low polydispersity index, e.g. a polydispersity index of lessthan 1.10, less 1.05, or less than 1.02, with respect to the compound.Polydispersity index, as used herein, means the ratio of weight averageto number average molecular weights. The composition can also bemonodisperse (i.e. have a polydispersity index of 1.00) with respect tothe compound. As will be appreciated from the disclosure herein,preparation of the composition such that the compound is presentpredominantly or entirely in one or a few particular molecular weightranges may provide advantages in terms of control of residence times ofthe compound in tissues (more rapid clearance of lower molecular weightcompounds) and viscosity of the composition (lower viscosity for lowermolecular weight compounds).

The composition can further comprise a protein that transfers heavychains of serum-derived proteoglycan bikunin to the compound. As one ofordinary skill will appreciate from this disclosure, co-administrationof the compound and such a protein to a site of inflammation may providefor more rapid and effective transfer of HC to the compound duringtherapeutic use, by supplementing the corresponding endogenous proteinat the site. The protein that transfers heavy chains of serum-derivedproteoglycan bikunin to the compound can be, for example, a mammalianTSG-6 protein, a human TSG-6 protein, a canine TSG-6 protein, a felineTSG-6 protein, and an equine TSG-6 protein. Such a protein may beobtained, for example, by purification from a natural source. Such aprotein may also be obtained, for example, in a recombinant form, e.g.based on expression and purification from a recombinant host or system.Such a protein may also be obtained, for example, by geneticengineering, e.g. by mutating or otherwise modifying a naturallyoccurring form of TSG-6 to form a novel version of TSG-6 thatnonetheless retains the enzymatic properties of TSG-6 protein. Methodsfor obtaining and/or modifying proteins as described above are wellknown in the art.

In another example aspect, a method of treating a site of inflammationin an individual in need thereof is provided. The method comprisesadministering to the site of the individual a therapeutically effectiveamount of a compound comprising the hyaluronan-containing structure:

the structure being as described above. Thus, for example, the methodcan comprise administering any of the compounds comprising the structureas described above.

In accordance with this method, the administering can comprise, forexample, administering a composition comprising the compound and apharmaceutically acceptable excipient by injection, inhalation, topicalrub, or ingestion. Thus, for example, the administering can compriseadministering any of the compositions comprising the compound and thepharmaceutically acceptable excipient as described above. Theadministering can be carried out once or multiple times, as needed, inorder to treat the inflammation.

The method can also further comprise administering, at the site, aprotein that transfers heavy chains of serum-derived proteoglycanbikunin to the compound, e.g. mammalian TSG-6 protein, recombinantmammalian TSG-6 protein, human TSG-6 protein, or recombinant human TSG-6protein, e.g. in a naturally occurring or recombinant form.

In accordance with this method, the therapeutically effective amount ofthe compound can be, for example, 0.3 to 8 mass equivalents ofpathological hyaluronan at the site, or 0.5 to 5 mass equivalents, 0.7to 3 mass equivalents, 0.8 to 2 mass equivalents, 0.9 to 1.5 massequivalents, or about 1 mass equivalent. The therapeutically effectiveamount of the compound can also be, for example, 2 to 7 mass equivalentsof pathological hyaluronan at the site, or 3 to 6 mass equivalents, 4 to5 mass equivalents, or about 4.5 mass equivalents.

The individual to be treated can be, for example, a mammal, such as ahuman, a dog, a cat, or a horse.

The inflammation at the site to be treated can be inflammation caused byan inflammatory condition such as, for example, rheumatoid arthritis,psoriatic arthritis, psoriasis, inflammatory bowel disease, or asthma.

In another example aspect, a method of decreasing heavy chainmodification of pathological hyaluronan at a site of inflammation in anindividual in need thereof is provided. The method comprisesadministering to the site of the individual a therapeutically effectiveamount of a compound comprising the hyaluronan-containing structure:

the structure being as described above. As will be appreciated, thismethod can also be carried out as described above, for example based onadministering any of the compounds comprising the structure as describedabove, e.g. by injection, inhalation, topical rub, or ingestion, e.g. byadministering any of the compositions comprising the compound and thepharmaceutically acceptable excipient as described above.

In another example aspect, a kit is provided. The kit comprises acompound comprising the hyaluronan-containing structure:

the structure being as described above. Thus, for example, the kit cancomprise any of the compounds comprising the structure as describedabove. The kit also comprises a pharmaceutical excipient, thepharmaceutical excipient also being as described above. Thus, forexample, the kit can comprise any of the compositions comprising thecompound and the pharmaceutically acceptable excipient as describedabove. The kit also comprises an instruction for use of the kit fortreating a site of inflammation in an individual in need thereof byadministering to the site a therapeutically effective amount of thecompound.

The kit can further comprise a protein that transfers heavy chains ofserum-derived proteoglycan bikunin to the compound, again e.g. mammalianTSG-6 protein, recombinant mammalian TSG-6 protein, human TSG-6 protein,or recombinant human TSG-6 protein, e.g. in a naturally occurring orrecombinant form. The individual can be, for example, a mammal, againe.g. a human, a dog, a cat, or a horse. The inflammation at the site canbe caused, for example, by an inflammatory condition, again e.g.rheumatoid arthritis, psoriatic arthritis, psoriasis, inflammatory boweldisease, or asthma.

In another example aspect, a kit is provided. The kit comprises acompound comprising the hyaluronan-containing structure:

the structure being as described above, and thus can comprise any of thecompounds comprising the structure as described above. The kit alsocomprises a pharmaceutical excipient, the pharmaceutical excipient alsobeing as described above, and thus can comprise any of the compositionscomprising the compound and the pharmaceutically acceptable excipient asdescribed above. The kit also comprises an instruction for use of thekit for decreasing heavy chain modification of pathological hyaluronanat a site of inflammation in an individual in need thereof byadministering to the site a therapeutically effective amount of thecompound. As will be appreciated, the kit can also be configured asdescribed above, e.g. to comprise a protein that transfers heavy chainsof serum-derived proteoglycan bikunin to the compound. The kit also canbe used for treatment of an individual being a mammal, e.g. a human, adog, a cat, or a horse. The kit also can be used for treatment of aninflammatory condition such as rheumatoid arthritis, psoriaticarthritis, psoriasis, inflammatory bowel disease, or asthma.

In another example aspect, a pharmaceutical composition is provided. Thecomposition comprises a compound comprising the structureA-(glcNAc*)_(x)-(glcA*-glcNAc*)_(n)-(glcA*)_(y)-B. The composition alsocomprises a pharmaceutically acceptable excipient. In accordance withthe composition, glcA* is D-glucuronic acid or substituted D-glucuronicacid, and glcNAc* is N-acetyl-D-glucosamine or substitutedN-acetyl-D-glucosamine, with the proviso that(glcNAc*)_(x)-(glcA*-glcNAc*)_(n)-(glcA*)_(y) includes at least onebinding site for TSG-6 protein. Also in accordance with the composition,A is selected from the group consisting of hydrogen, a substituent thatdoes not comprise a binding site for TSG-6 protein, a substituent thatinterferes with binding of TSG-6 protein immediately adjacent thereto,and chondroitin. Also in accordance with the composition, B is selectedfrom the group consisting of hydroxyl, a substituent that does notcomprise a binding site for TSG-6 protein, a substituent that interfereswith binding of TSG-6 protein immediately adjacent thereto, andchondroitin. Also in accordance with the composition, x=0 or 1, y=0 or1, and n=3 to 70, with the proviso that if n=3, then at least one of xand y=1. The composition is suitable for administration by injection,inhalation, topical rub, or ingestion.

As one of ordinary skill will appreciate, like the compound comprisingthe hyaluronan-containing structure:

the compound comprising the structureA-(glcNAc*)_(x)-(glcA*-glcNAc*)_(n)-(glcA*)_(y)-B can also be used inthe methods and kits disclosed herein, based on irreversible transfer ofHC thereto. Based on the disclosure herein, one of ordinary skill wouldbe able to identify and confirm any of a wide range of compoundscomprising the structureA-(glcNAc*)_(x)-(glcA*-glcNAc*)_(n)-(glcA*)_(y)-B for use in suchmethods and kits without undue experimentation or effort.

In another example aspect, controlled and extended release of the LMW HAoligosaccharides in tissues subject to or experiencing inflammation maybe achieved through encapsulation in tyramine-based (TB) HA hydrogels.Such gels are described in detail in U.S. Pat. Nos. 6,982,298,7,368,502, 7,465,766, 8,021,350, 8,080,260, 8,137,688, and 8,138,265,the contents of which are incorporated herein by reference. LMW HAencapsulated in TB-HA hydrogels would be released at a rate predictedbased on the size of the LMW HA and the porosity (concentration) of theHA in the TB-HA hydrogel, making the LMW HA accessible to TSG-6 enzymefor quantitative transfer of HC thereto. Such gels, implanted at aninflammation site, may also be broken down over time via the naturalaction of native hyaluronidase or free radicals, thus further releasingencapsulated LMW HA, making the latter accessible to TSG-6 enzyme forquantitative transfer of HC thereto. Alternatively, an implanted HAhydrogel itself may serve as a supply for LMW HA, whereby hyaluronidaseor free radical mediated cleavage of HA chains in the gel releasessmaller HA chains of appropriately low molecular weight, such that thereleased LMW HA chains are effective to irreversibly transfer HCattached thereto via the action of TSG-6 in vivo.

In another example aspect, a periodically-substituted HMW HA can be usedto supply LMW-HA domains that will function as LMW HA in the disclosedmethods. For example, tyramine-substituted HMW HA molecules (e.g., thesame or similar molecules used to prepare the aforementioned TB-HAhydrogels in the above-incorporated patents) have tyramine adductssubstituted onto the HA backbone at spaced or periodic locations alongthe macromolecular HA length. For example, to prepare the TB-HAhydrogels mentioned above HMW HA molecules having a periodicity oftyramine substitution of about 1 tyramine adduct every ˜40monosaccharides is preferred. The periodic spacing of tyramine adductspresents a series of discrete LMW-HA domains along the length of the HMWHA macromolecule, wherein each of the LMW-HA domains is accessible toTSG-6 enzyme for quantitative transfer of HC thereto. Thus, according tothis embodiment a HMW HA macromolecule can be prepared that provides aseries of LMW-HA domains separated by period adduct species where theadduct will not be recognized by the TSG-6 enzyme as native HA, andwhich will accordingly prevent or inhibit binding of that enzyme tomediate HC transfer from the LMW-HA domain. These domains thus will actlike LMW HA if the non-native adducts (e.g. tyramine) are substituted onthe HMW HA backbone with an appropriate periodicity to generate LMW-HAdomains that serve as irreversible acceptors for HC transfer. As will beappreciated, the tyramine-substitution rate can be selected to providethe desired level of periodicity to correspond to the desired LMW-HAdomain length (e.g. about every 10, 15, 20, 25, 30, 35 or 40monosaccharides), or some other length below which the resulting LMW-HAdomains will function as irreversible acceptors for HC transfer asdescribed above. Methods to substitute tyramine onto HA, as well as tocontrol the substitution rate, are disclosed in the aforementionedpatents incorporated by reference. While tyramine is described here asthe non-native HA adduct for providing LMW-HA domains, it will beappreciated that other non-native adducts that will not be recognized asnative HA by the TSG-6 enzyme can be used.

As will be appreciated from the foregoing, there are at least two modesby which HMW HA can be used to provide a source of LMW-HA domains usefulto irreversibly transfer HCs based on the disclosed methods. One mode isto supply a hydrogel that is prepared by cross-linkingperiodically-substituted moieties along the length of HMW HA (e.g. theTB-hydrogels disclosed in the aforementioned patents), wherein thesubstitution rate is such that upon breaking down (e.g. digestion viahyaluronidase or free radicals) the hydrogel at an inflammation site,the resulting HA fragments have sufficiently low molecular weight tofunction as LMW HA in the disclosed methods. A second mode is to supplya periodically-substituted HMW HA macromolecular species not in across-linked or hydrogel form, but as free macromolecular strands withperiod adducts that define LMW-HA domains consisting of ‘naked’ HAbetween adjacent adducts. These LMW-HA domains behave as LMW HA toirreversibly transfer HCs via a TSG-6-mediated reaction as discussedabove. Both modes can be used to irreversibly quantitatively transferHCs from tissue HA to LMW HA or LMW-HA domains introduced to the site ofinflammation via injection therapy or some other method, therebyreducing or inhibiting the inflammatory process.

It should be noted that in the free-strand modality discussed above,digestion of the free strands is also possible at an inflammation site.However, such digestion will produce LMW HA fragments correspondingsubstantially to the LMW-HA domains in the substituted HMW HA molecule.Accordingly, these fragments still will function to irreversibly acceptHCs transferred from tissue HA according to the disclosed methods.

In addition to treating inflammation, an alternative embodiment involvesusing LMW HA oligosaccharides as a female contraceptive. The TSG-6mediated transfer of HC from IαI to HA is required for female fertilityas demonstrated by the fact that knock-out mice for either TSG-6 orbikunin, a component of IαI, are infertile. If the LMW HA could be usedto prevent HC transfer to the HA in the ECM of the developing cumuluscell-oocyte complex, oocyte maturation could be disrupted and oocytefertilization and pregnancy prevented. This embodiment provides anon-hormone-based therapy for contraception. The use of LMW HA as acontraceptive as here described may have lower potential for sideeffects and other risks associated with conventional, hormone-basedtherapies.

It has been observed that tyramine-substituted (TS) HA can be modifiedwith HC in vitro via recombinant TSG-6 and serum as a source of IαIsimilar to “naked” HA, and that the resulting HC-decorated TS-HA(hereafter HC-TS-HA) can be subsequently cross-linked with horseradishperoxidase and hydrogen peroxide as described in the above-incorporatedpatents to form tyramine-cross linked hydrogels of the HC-TS-HA (suchhydrogels being referred to herein as HC-TB-HA). These HC-TB-HAhydrogels can be used for various tissue engineering and repairapplications wherein the gels are engineered to be patient-specificthrough the use of a patient's own serum as the source of HC-bikunin. AsHC transfer to HA has been implicated in induction of angiogenesis,these HC-TB-HA hydrogels may be useful in those applications in whichinduction of blood vessels around implanted hydrogel is desirable. Forexample, in applications involving embedding of cells/tissues inimplanted hydrogel constructs (i.e., hydrogel constructs containing fatfor facial reconstruction, islet cells as a synthetic pancreas fortreatment of diabetes, or ovary tissue as a synthetic ovary). Formationof a capillary bed around the hydrogel construct is desirable as asource of nutrients to feed embedded cells maintaining cell viability.

The disclosed methods and compositions are based on our recent discoverythat in the presence of TSG-6, LMW HA of defined size irreversiblydisplace HCs, a component of the serum-derived proteoglycan, IαI, fromHMW HA. The displaced HCs are irreversibly transferred to the LMW HA byaction of the TSG-6 enzyme. This provides a paradigm for quantitativelyremoving pathologic HC ligands from HA of inflamed tissues, potentiallyalleviating or eliminating inflammation. This will provide not onlypatient relief from the symptoms of inflammatory disease, butpotentially a treatment to prevent or inhibit progression of theinevitable joint (tissue) damage caused by the disease.

A feature of the disclosed methods is that their therapeutic effect hasthe potential for fewer and less severe side-effects compared to currentmedications and interventions. In particular, current medications andinterventions either treat only the signs and symptoms of the disease,or are designed to slow progression of the disease through modificationof immune cell production or function. As expected, these medicationshave many undesirable side effects including risk of life threateninginfections, hematologic issues, kidney and liver damage, severeimmunosuppression, death, and long-term effects such as an increasedrisk of cancer. Our recent discovery of a mechanism whereby thepathological HC-HA matrix in inflamed synovial joints can be restored toa normal, non-inflammatory state through HC transfer to LMW HA providesa mechanistic approach for novel therapeutics and therapies to reducethe signs and symptoms as well as halt the progression of irreversiblejoint damage in patients by targeting the activating signal for theseimmune cells rather than the cells themselves.

Conventional medications for treating inflammation work globally toexert their effect directly on a patient's immune cells affecting thepatient's inflammatory pathways. As a result, such medications areassociated with unwanted side effects such as life threateninginfections, hematologic complications such as leucopenia or anemia, andincrease risk of malignancy. Conversely, the mode of action for the LMWHA is expected to be more localized and indirect (i.e. not directlyaltering immune cell function) via removal of the specific matrix signalresponsible for attracting and activating immune cells at theinflammatory site. As a result, the use of LMW HA in the treatment ofinflammatory disease as disclosed herein may be expected to have lowerpotential for (and less severe) side-effects compared to conventionaltreatments.

While the foregoing discussion has been related to HA, it bears notingthat materials other than HA may be used to mediate the transfer of HCs,from tissue HA as a treatment for inflammation. For example, it is knownthat HCs can be transferred by TSG-6 to un-sulfated chondroitin sulfatechains (0S-CS), which have a disaccharide repeat identical to HA exceptfor the conformation of one hydroxyl group. While HA is a betteracceptor of HCs from TSG-6 than 0S-CS, the 0S-CS works and presumablywould show a similar size dependence relative to the reversibility ofthe reaction. It may also be that HC-0S-CS is not recognized by immunecells as this is not a natural product driving the immune process.Sulfated CS is reported not to be acceptors for HC transfer by TSG-6,but this could be dependent on the type of sulfation pattern, as the CSchain that is part of bikunin and IαI has both sulfated and un-sulfateddisaccharides and does act as a HC acceptor/donor. Other molecules likeheparin, heparan sulfate, and alginate may also be possible substitutesfor HA or any other molecule containing hexuronic acid residues.Finally, chimeras of glycosaminoglycan chains composed of stretches ofHA interrupted with stretches of other glycosaminoglycans such aschondroitin, chondroitin sulfate, heparin, or heparan sulfate could beused to create LMW HA domains within HMW glycosaminoglycan chainssimilar to that described above for the periodic substitution of HMW HAby adducts such as tyramine.

EXAMPLES Example 1

Rheumatoid Arthritis (RA) is a chronic, systemic inflammatory disorderaffecting more than 1% of the world population, and about 2 millionpeople in the US alone. RA may affect many tissues and organs, butprincipally attacks synovial joints (SJs). The process produces aninflammatory response of the synovium (synovitis) secondary tohyperplasia of synovial cells, excess synovial fluid (SF), thedevelopment of pannus in the synovium, and invasion by white bloodcells, which produce a variety of tissue destructive factors. Thepathology of the disease process often leads to the destruction ofarticular cartilage and ankylosis of the joints, a disabling and painfulcondition, which can lead to substantial loss of function and mobilityif not adequately treated. Currently, the cause of RA is unknown, andthere is no cure with the only option at end stage disease being totaljoint replacement. Current medications and interventions either treatonly the signs and symptoms of the disease, or are designed to slowprogression of the disease through modification of immune cellproduction or function. As expected, these medications have manyundesirable side effects including risk of life threatening infections,hematologic issues, kidney and liver damage, severe immunosuppression,death, and long-term effects such as an increase risk of cancer. Ourrecent discovery of a mechanism whereby the pathological HC-HA matrix ininflamed SJs can be restored to a normal, non-inflammatory state throughHC transfer to LMW HA provides a mechanistic approach for noveltherapeutics and therapies to reduce the signs and symptoms as well ashalt the progression of irreversible joint damage in RA patients.

We have learned that the formation of the pathological HC-HA complex isa dynamic process whereby TSG-6 readily “swaps” HCs along the HMW HAbackbone until reaching equilibrium (FIG. 2A). Importantly, although LMWHA readily serves as an acceptor for HC transfer from HMW HC-HA, it isunable to serve as a HC donor (FIG. 2B) resulting in irreversibletransfer of HCs from HMW to LMW HA. Thus, smaller fragments of HA canserve as the basis for anti-inflammatory therapies whereby thepathological HC-HA matrix in inflamed SJ can be restored to a normal,non-inflammatory state via irreversible TSG-6-mediated transfer of HCsfrom ECM-nascent HMW HA to LMW HA strands. This is the first mechanisticdescription of a function for LMW HA, which provides the basis fortherapies for inflammatory diseases via LMW HA supplements. Unlikecurrent medications, which act directly through modification of immunecell production or function, LMW HA therapy may provide a saferalternative for RA treatment as well as halt the progression ofirreversible joint damage in these patients without the unwanted sideeffects of existing treatments.

Abnormal Heavy Chain Modification of Hyaluronan in Synovial Fluid inRheumatoid Arthritis.

Hyaluronan (HA) is a large glycosaminoglycan (GAG) in which thedisaccharide (glucuronic acid-β1,3-N-acetylglucosamine-β1,4-) isrepeated several thousand times (4). It is synthesized at the cellsurface of type B synoviocytes, reaching concentrations of 3-4 mg/ml inthe SF (5). Its unique physical properties as a high molecular weight(HMW), anionic, structural carbohydrate confer its ability to functionas a hydrated, viscoelastic lubricant for the SJ. Under normalconditions, it exists as a “naked” glycosaminoglycan, lacking a covalentbond to any protein. During joint inflammation, this relatively inert HAmatrix is transformed into a ligand for inflammatory cell receptors viathe covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI)via TSG-6 protein.

This has been shown experimentally, as follows (FIG. 10). Synovial fluid(SF) and serum from a patient with rheumatoid arthritis (RA) weretreated +/−hyaluronidase (hyase) and analyzed by Western blot, probingthe blot with a polyclonal antibody that has reactivity against theheavy chains (HC) and bikunin of the tri-peptide inter-α-inhibitor(IαI). Hyaluronidase treatment of SF released heavy chains bound tohyaluronan so that they ran as a distinct 83 kDa band on the gel. Thisdid not occur when serum was treated with hyaluronidase, confirming theabsence of the HC-HA complex in this patient's serum. In serum, heavychains (83 kDa) are linked to bikunin (37 kDa) via a chondroitin sulfateglycosaminoglycan to form a tri-peptide (IαI) or a di-peptide (pre-IαI).

In 1965, Sandson, et al, first reported that HA from pathological SF wasassociated with the components of IαI (6). In 1999, this association wasidentified as a covalent bond between the HCs of IαI and HA (7). IαI isa serum proteoglycan, synthesized by hepatocytes in the liver, andsecreted into the circulatory system where it reaches relatively highconcentrations (0.15-0.5 mg/ml) (8). It is composed of 3 polypeptides:the trypsin inhibitor called bikunin, (16 kDa) and two HCs (−83 kDaeach) (9). The two HCs are covalently attached to bikunin's singlechondroitin sulfate (CS) chain by an ester linkage between a HCaspartate and the 6-OH of galNAc in the CS. Under normal conditions, IαIis absent from SF since the synovial barrier prevents proteins largerthan 100,000 kDa from entering the synovial space (6), but duringinflammation, IαI leaks into the synovial space through dilatedcapillaries where it serves as a HC donor for the transfer of HCs to HAvia the enzyme TSG-6.

TSG-6 is a 35 kDa protein that is synthesized and secreted by many cellsafter treatment with tumor necrosis factor alpha (TNFα) and interleukin1 (10). TSG-6 binds to HA via its link module and can form a HC-TSG-6complex with the HCs of IαI (FIG. 1) (11). Through our investigations,and others, we identified TSG-6 as the enzyme responsible for thecovalent transfer of HCs to HA (12-14). Elevated levels of TSG-6 havebeen observed in asthmatic bronchoalveolar lavage fluid (15), in theairway epithelium and secretions of smokers (15), and in infarctedregions following stroke (16). The covalent transfer of HCs from IαI viaTSG-6 transforms the HA matrix into a ligand for inflammatory cellreceptors. This was illustrated in a study which isolated the HC-HAcomplex from inflamed SF and showed that leukocytes preferentially boundto the HC-HA complex when compared to HA alone (17). Similarly, our ownstudies have shown that the artificial addition of HCs to HA “cable”structures via the addition of recombinant TSG-6 significantly enhancedleukocyte adhesion (not shown). The white blood cell count from inflamedSF are 10-500 fold above normal levels, driving inflammation in thisjoint. The HC-HA complex functions as a specific ligand for theseinflammatory cell receptors, providing a “sticky” platform for adhesionwithin this viscoelastic fluid. The resolution of inflammation in RA andother inflammatory conditions should, at least partially, involve arestoration of the HA matrix in SF to its original state, unmodifiedfrom HCs.

Irreversible Transfer of Heavy Chains to Low Molecular Weight Hyaluronanvia TSG-6.

We have discovered a mechanism whereby LMW HA could function in theclearance of HCs from HC-HA matrices in SF. This involves a uniqueability for LMW HA to serve as an irreversible HC acceptor from HMW HAvia transfer by TSG-6. Results were as follows.

A preliminary range study for the irreversible transfer of heavy chainsto hyaluronan oligosaccharides revealed molecular weight gel shifts ofheavy chains (HCs) transferred from inter-α-inhibitor (IαI) and pre-IαIto hyaluronan (HA) and swapping between HA molecules to HAoligosaccharides (FIG. 7). Results from Western blots with mouse serumshow that HA6 (and presumably <HA6) are not HC acceptors or donors, thatHA8-14 can serve as HC acceptors but not donors, and that HMW HA (˜1500kDa) can be both a HC acceptor and donor (FIG. 7A). Results with humanserum show that HA having about 160 monosaccharide (˜30 kDa HA)functions like HMW HA (˜1500 kDa) as both a HC acceptor and donor,unlike HA8 which is only a HC acceptor (FIG. 7B). These data establishHA8 as the lower HA (composed of complete disaccharide units) size limitfor irreversible HC transfer and show that the upper limit is belowabout 30 kDa HA. These experiments were repeated 2 times.

An additional study revealed irreversible transfer of heavy chains fromHMW HA to LMW HA via TSG-6 (FIG. 8). Results from a Western blot showthe molecular weight gel shift of HCs transferred from inter-α-inhibitor(IαI), pre-IαI, or HMW HA to LMW HA (FIG. 8A). Quantification of the HCband from the Western blot is also shown, in which error bars representstandard deviation (n=3) (FIG. 8B).

Considering FIG. 8A in more detail, lanes 1-3 show that HCs are presentin serum as part of the IαI or pre-IαI complexes. This is unaffected bythe addition of LMW HA 10 monosaccharides long (HA10) (lane 2) or by theaddition of a HMW HA ˜1600 kDa (lane 3). In lane 4, serum supplementedwith TSG-6 results in release of free HCs from IαI and pre-IαI.Supplementation of this serum with additional LMW HA (i.e. HA10) in thepresence of TSG-6 causes a gel shift of the HC from IαI and pre-IαI(comparing lane 5 with 4). When the serum is supplemented with HMW HAinstead of LMW HA in the presence of TSG-6 (lane 6), most of the HC failto enter the gel because their attachment to HMW HA makes the HC-HAcomplex too large for resolution on this gel. In lane 7, HC from serumwere transferred to HA10 for the first 4 hrs and then the reactionmixture was spiked with equivalent amounts of HMW HA for the remaining24 hrs (48 hrs total). Since the HC gel shift is identical to theaddition of HA10 alone (as seen in lane 5), this implies that oncetransferred to HA10, the majority of HC remain attached to this HAoligosaccharide even in the presence of HMW HA. However, the reverse ofthis does not occur (lane 8). In this instance, HC were transferred toHMW HA in the first 24 hrs, and then the reaction mixture was spikedwith equivalent amounts of HA10 for the remaining 24 hrs (48 hrs total).This resulted in a HC gel shift identical to lane 7, implying that theHC were transferred from HMW HA to HA10 in an irreversible manner.Similarly, when serum was supplemented with HMW HA and HA10 in thepresence of TSG-6 simultaneously for 48 hrs (lane 9), the majority of HCdemonstrated a gel shift indicative of transfer to HA10 alone. Thesedata demonstrate that TSG-6 “swaps” HCs between HA molecules in a sizedependent manner which is reversible for HMW HA, but irreversible forLMW HA, proving that LMW HA can function as an irreversible acceptor forHC derived from HMW HC-HA.

Accordingly, we have learned that TSG-6 readily “swaps” HCs between HAmolecules of HMW HA. Although LMW HA can serve as a HC acceptor, it isunable to serve as a HC donor, thereby making the reaction irreversible.This can be explained in the following model. In order for HC transferto LMW HA to occur, the TSG-6-HC intermediate must bind LMW HA via theHA binding “link” domain of TSG-6 and transfer the HC to LMW HA. Withoutwishing to be bound by theory, it is believed that once the HC istransferred, steric hindrance by the attached HC prevents TSG-6 frombeing able to bind the HA portion of LMW HC-HA, thereby preventingremoval of its HC (FIG. 3).

Evidence that this process can be applied to human SF to remove HCs fromHMW HA via irreversible transfer to LMW HA includes the following (FIG.11). A Western blot of SF probed with an antibody that binds only heavychains (HCs) is shown. Free HC, or HC bound to LMW HA, is present in SFfrom an RA patient (lane 1). When LMW HA containing a HA fragment of 10monosaccharides (HA10) was applied to this SF, endogenous TSG-6transferred HCs from HMW HC-HA onto HA10 (compare lanes 1 and 2),causing a ˜1.6 kDa shift higher in MW. When both exogenous TSG-6 andHA10 are applied to the SF (lane 4), HCs from HMW HC-HA are transferredto HA10 in an irreversible manner. This induced a 1.6 kDa vertical gelshift similar to lane 2, albeit the intensity of the band is strongerwith the addition of exogenous HA10. Treatment of SF with hyaluronidase(hyase) released all the HCs bound to HMW HA (lane 5), showing that theaddition of more HA10, or more time, would be required to completelyremove all of the HCs from HMW HA. These data demonstrate a mechanismwhereby HCs are removed from HMW HA, and transferred to LMW HA, in SFfrom RA patients.

Role for Low Molecular Weight Hyaluronan in the Removal of Heavy Chainsfrom High Molecular Weight Hyaluronan in the Synovial Fluid ofRheumatoid Arthritis Patients.

In the synovial cavity, HA degradation into smaller fragments is mostlikely to occur via hyaluronidase digestion by phagocytic cells (i.e.macrophages) and by oxidative damage via reactive oxygen species(released by neutrophils) (18). We now know that these fragments havethe ability to serve as irreversible acceptors of HCs derived from HMWHC-HA (FIG. 8).

In addition to the LMW HA fragments that are locally produced as adegradation product of HMW HA in SF, our data suggest that serum fromtested RA patients contained relatively high levels of circulating LMWHA (FIG. 12). Specifically, synovial fluid (SF) from four patients (a-d)was treated +/− with hyaluronidase and the amount of released HCsanalyzed by Western blot (panel A). Serum from these same patients wastreated +/− with TSG-6 (panel B). This transferred HCs from IαI ontocirculating LMW HA, or alternatively, TSG-6 released free HCs. Free HCand HC bound to LMW HA (doublet above the main HC band) were present inall SF (panel A). Hyaluronidase treatment of SF released HCs bound toHMW HA. HMW HC-HA was also observed in serum as a smear at the top ofthe gel (panel B; notice patients a & d). This was not seen in the SFbecause the HMW HA in SF is apparently larger than in serum. Treatmentwith TSG-6 caused a strong gel shift of the HCs from IαI with patient“b” to form a HC smear indicative of transfer to LMW HA. This also wasprominent with patients “c” and “d.” TSG-6 treatment of patient “a”removed HCs from HMW HA to LMW fragments. Furthermore, the appearance ofa HMW HC-HA smear with patient “c” implies that there was circulatinglevels of “naked” HMW HA in this patient. These data show that the HCsare bound to both HMW and LMW HA in SF of RA patients, with HMW HC-HApredominating. Relatively high levels of LMW HA were found in the serumfrom RA patients, which could serve as an irreversible acceptor of HCsfrom HMW HC-HA.

Based on the above, we propose a therapy whereby LMW HA is administeredlocally at the site of inflammation (with or without exogenous orendogenous TSG-6 enzyme and/or IαI) to serve as an irreversible acceptorfor HCs and a modulator of the pathological HC-HA complex in the SF ofinflamed SJs. Note that exogenous or endogenous TSG-6 and/or IαI couldbe supplied in a bolus together with the LMW HA to supplement the enzymeand IαI already present at the site, or to provide those species in aninflammation site where they may be naturally deficient.

The Size Range Whereby LMW HA can Function as an Irreversible Acceptorof HCs.

HMW HA can both accept and donate HCs as they are “swapped” between HAmolecules via the enzyme TSG-6 (FIGS. 2B and 7). Interestingly, a HAfragment of 10 monosaccharides (HA10) was able to accept HCs from HMW HAvia TSG-6, but once bound, the enzyme was unable to use HC-HA10 as a HCdonor (e.g. FIGS. 8 and 11). Moreover, HA molecules having between 8 and14 monosaccharide units have been found to perform suitably as acceptorsfor TSG-6-mediated HC attachment while not permitting the reversereaction to remove HCs from the HA molecules (via TSG-6) once attached.While HA8-14 molecules have performed suitably in this regard to provideirreversible attachment of HCs transferred via TSG-6 from HMW HA, lowermolecular-weight HAs having six or fewer monosaccharide units did notwork in our experiments. Without wishing to be bound by theory, it isbelieved that these small HA molecules did not possess sufficient lengthto accommodate a TSG-6 binding site necessary to initiate TSG-6-mediatedHC-transfer to the HA molecule. Similarly, based on our theory of sterichindrance-irreversible HC-transfer from HMW to LMW HAs, suggests therewill be a threshold HA length beyond which the molecule will havesufficient length to accommodate a TSG-6 binding site while HCs areattached. At this threshold length, the HA will no longer serve as aneffective LMW HA in the disclosed methods and therapies forquantitatively transferring HCs from ECM-nascent HMW HA. Our experimentssuggest that this threshold HA length is about 160 monosaccharide unitsor greater. The LMW HA molecules used in the disclosed methods andtherapies preferably has a length in the range of about 8-25monosaccharide units, 8-20 monosaccharide units, 8-15 monosaccharideunits or 8-10 monosaccharide units. Assigning a biological function to adistinct range of HA sizes may be relevant in the treatment of differentinflammation syndromes in different tissues because the size of HAvaries by tissue type and stage of inflammation (18).

Clearance of LMW HA and HC-HA in the Synovial Fluid.

SF is not a static pool, but is continually being absorbed andreplenished by the synovial lining of the joint cavity (19). Raw SF isformed as plasma diffuses from synovial capillaries, into theinterstitial matrix, and across the fenestrated membranes of type Bsynoviocytes into the synovial cavity. Subsequently, synoviocytessecrete macromolecules such as hyaluronan and the proteoglycan lubricininto the raw serum exudate. SF is absorbed back through the fenestratedmembranes and the interstitial matrix of the synovium and into thesynovial venous and lymphatic system where it is returned to thecirculatory system to be cleansed by the liver and kidneys (20).

Several studies have shown that there is a disparity between theturnover of macromolecules in SF, implying selective permeability of thesynovial membrane (3, 21-22). For example, the turnover of water andalbumin in rabbit and human knees is about 1-2 hr (21-22). In contrast,the half-life of HMW HA is an order of magnitude slower, ranging from15-28 hrs (22-24). The turnover of HA in SF is a function of its size,with lower molecular weights presenting shorter half-lives (3). Sincethe covalent transfer of HCs (83 kDa) to HMW HA in SF duringinflammation would increase its accumulative MW, we predict that theturnover of HMW HC-HA would be even longer than naked HMW HA alone. Incontrast, we expect that the turnover of HA10 (1.6 kDa) and HC-HA10 (85kDa) in SF would resemble that of water and albumin (67 kDa). These areimportant distinctions because they could explain the means whereby thepathological HC modification of HMW HA could be flushed out of SF viatheir irreversible transfer to LMW HA.

Of note, while previous studies have shown that HMW HA is rapidlycleared from the circulatory system via the liver (20), this may not betrue for LMW HA in the size range that can function as an irreversibleacceptor for HCs and the effect that the HC modification might have onthis process is unknown.

Summary.

We have identified a novel mechanism whereby LMW HA can function as anirreversible acceptor to restore HC modified matrices in inflamed SJs totheir original state.

Example 2 Irreversible Transfer of Heavy Chains to LMW HA Via TSG-6

Conventional RA therapies, such as corticosteroids and disease-modifyinganti-rheumatic drugs (DMARDS) treat only the signs and symptoms of thedisease, or slow its progression through modification of immune cellproduction or function. These treatments globally affect the immunesystem with serious long-term side effects. In contrast, thecompositions and methods disclosed herein rely on a mechanism wherebyrelatively small HA molecules can be used to remove HCs from HC-HAmatrices in SF, thereby restoring them to their normal (naked) state.This technology is based upon the observation that while TSG-6reversibly swaps HCs between large HA molecules found in SF, HC swappingto smaller HA molecules is irreversible. In other words, while large HAmolecules are both HC acceptors and donors, smaller HA molecules areonly HC acceptors.

For example, results from a Western blot show irreversible HC “swapping”from HA1000K (high molecular weight HA with a molecular weight of 1000kDa, also termed HA_(1000kDa)) to HA14 (HA oligosaccharide containing 14monosaccharides) via recombinant TSG-6 (FIG. 13). The HCs of IαI fromhuman serum (lane 1) are completely transferred to HA14 (lane 2) and toHA1000K (lane 3) in 24 hours. Note that HCs transferred to HA1000K donot enter the gel because the HC-HA complex is too large, thus givingthe appearance that they disappeared. In lane 4, HCs were firsttransferred to HA1000K for 24 hours, after which the reaction mixturewas spiked with HA14 to allow swapping of HCs from HA1000K to HA14 for72 hours. In lane 5, the reverse sequence was applied (i.e. transfer ofHCs to HA14 for first 24 hours followed by incubation for 72 hours withHA1000K for 72 hours). Notice that while the HCs were able to swap fromHA1000K to HA14 (lane 4) the reverse of this did not occur (lane 5).Thus, transfer of HCs to HA14 is an irreversible event. This is furtherillustrated in lane 6 in which both HA1000K and HA14 were appliedsimultaneously for 72 hours. Thus, while HA1000K is both a HC acceptorand donor, HA14 is exclusively an irreversible HC acceptor. Similarresults were obtained with mouse serum.

This phenomenon can be exploited to remove HCs from pathological HC-HAmatrices found in RA and other inflammatory diseases, thereby making HAless sticky by removing the HC-HA ligand by which inflammatory cellsadhere to HA matrices (FIG. 14). Thus, for example results from aWestern blot show irreversible transfer of heavy chains from mousesynovial fluid hyaluronan to hyaluronan oligosaccharides, includingevidence for irreversible HC transfer from HMW HC-HA to HA14oligosaccharides as well as evidence of endogenous SF TSG-6 activity inSF derived from mice subjected to RA is provided. In lanes 1-2, SF wastreated with and without hyaluronidase for 72 hours. In lane 4, in thepresence of exogenous recombinant TSG-6, HCs from HMW HC-HA wereirreversibly transferred to HA14 oligosaccharides. In lane 3, in theabsence of exogenous TSG-6, HC transfer to HA14 occurred as a result ofendogenous SF TSG-6 activity. Similar results were obtained with humanSF derived from RA patients (not shown).

Example 3 Size-Range Study of Irreversible Heavy-Chain Transfer I

We have learned that the size range for irreversible HC transfer to HAis from 8 monosaccharides (corresponding to about 1.5 kDa) to at least87 monosaccharides (corresponding to about 16 kDa). Evidence includesthe following (FIG. 15). Recombinant TSG-6 covalently transferred HCsfrom serum derived IαI (lane 1) to different sizes of HA polysaccharides(lanes 2-9) for 4 hrs at 37° C. to form the HC-HA complex with thesepolysaccharides. Afterwards, HA1000K was added to an aliquot of thesesamples for 24 hrs (+) (lanes 3,5,7,9), or HA1000K was not added (−)(lanes 2,4,6,8). In this experiment, the HAs ranged in size from HA14(i.e. 14 monosaccharides, corresponding to 2.66 kDa) (lanes 2,3) to HA87(i.e. 87 monosaccharides, corresponding to 16.48 kDa) (lanes 8,9). Thedata show that once HCs were transferred to these HAs ranging from 3-16kDa, TSG-6 could not remove the HCs to transfer them to HA1000K.

Actual numbers for HA molecular weight and calculated monosaccharidesbased on the MALLS-SEC analysis by Hyalose are provided (FIG. 15).Quantitation of this gel indicated that the (+) bands were approximately20% less than the (−) bands for HC-HA_(2.66kDa), HC-HA_(7.05kDa), andHC-HA_(10.70kDa), and approximately 40% less for HC-HA_(16.48kDa). Thusthe 4 hour incubation required to transfer all of the HCs from the IαIto the HA polysaccharides was insufficient to allow all of the HC-HAformed to be at irreversible sites on the HA polysaccharides.

When the initial transfer of HCs from the IαI to the HA polysaccharidesis allowed to incubate for 24 hrs at 37° C. before addition of thecompeting polysaccharide (HA1000K), it appears all of the HC transferhas shifted to irreversible sites on the polysaccharides (FIG. 16).Specifically, recombinant TSG-6 covalently transferred HCs from humanserum derived IαI (lane 2) to different sizes of HA polysaccharides(lanes 3-10) for 24 hrs at 37° C. to form the HC-HA complex with thesepolysaccharides. After this initial 24 hrs, HA1000K was added to analiquot of these samples for 24 hrs (+) (lanes 4,6,8,10) or HA1000K wasnot added (lanes 3,5,7,9). The data show that once HCs were transferredto HA57 (i.e. 57 monosaccharides, corresponding to 10.70 kDa) (lanes3,4,7,8) or HA87 (i.e. 87 monosaccharides, corresponding to 16.48 kDa)(lanes 5,6,9,10), TSG-6 could not remove the HCs to transfer them toHA1000K (HC-HA1000K is too large to enter the gel).

A further experiment also showed that HA8 to HA21 can serve asirreversible HC acceptors (FIG. 17). Specifically, recombinant TSG-6covalently transferred HCs from human serum derived IαI (A,B: lane 2) todifferent sizes of HA polysaccharides (A,B: lanes 3-9) for 2 hrs at 37°C. to form the HC-HA complex with these polysaccharides. After thisinitial 2 hrs, HA1000K was added to an aliquot of these samples for 24hrs (+) (A,B: lanes 3,5,7,9) or HA1000K was not added (+) (A,B: lanes2,4,6,8). The data show that once HCs were transferred to HA containing8, 10, 12, 14, 18 and 21 monosaccharides (A: lanes 6,7; A: lanes 8,9; B:lanes 2,3; B: lanes 4,5; B: lanes 6,7; B: lanes 8,9; respectively),TSG-6 could not remove the HCs to transfer them to HA1000K (HC-HA1000Kis too large to enter the gel). The HA4 and HA6 data are complicated. Inthe absence of any HA of a size that can accept HCs, i.e. HA4 and HA6,TSG-6 is able to slowly, but quantitatively, release free HCs over 24hrs. For the HA4 and HA6, the small amount of free HC band seen uponaddition of HA1000K after the initial 2 hr incubation is the result offree HCs generated by TSG-6 in the first 2 hrs in the absence of HAacceptor when no HA1000K is present. The remainder of IαI HC istransferred to the HA1000K over the next 24 hrs. Thus, it is believedthat HA polysaccharides from 1.53 to 16.48 kDa (HA8 to HA87) serve asirreversible HC acceptors.

Based on the foregoing, we have determined that HA polysaccharides from1.53 to 16.48 kDa (HA8 to HA87) are irreversible HC acceptors.

Moreover, as noted above, we have also determined that transfer of HCsto HA that is 30.6 kDa is reversible. Thus, the transition betweenreversibility and irreversibility lies between HA that is 16 and 30.6kDa.

Example 4 Heavy Chain Isotypes

Five different homologous HCs (HC1-5) are present in two gene clustersmapped to different chromosomes (HC1, 3 and 4 to chromosome 3 and HC2and 5 to chromosome 10). HC4 is unique in that it is not part of IαI, isnot transferred to HA, and is secreted as a free HC. We have shown thatIαI in mice and/or humans is comprised of at least 3 different HCisotypes (HC1, HC2 and HC3) (FIG. 18 shows this for HC1 and HC2; HC3 wasalso confirmed, but not shown).

Specifically, Western blots showing the kinetics of HC1 (A) and HC2(B,C) transfer from human (A,B) and mouse (C) serum-derived IαI andpre-IαI to HA8 (eight monosaccharides long) via the addition ofrecombinant TSG-6 are provided (FIG. 18). Transfer of HC1 from IαI inhuman serum to HA8 was relatively rapid (2-4 hrs), and did not result inthe accumulation of the pre-IαI intermediate (panel A). In contrast,transfer of HC2 from IαI in human serum to HA8 was relatively slow (≧24hrs), and resulted in the accumulation of the pre-IαI intermediate(panel B). Similarly, transfer of HC2 from IαI and pre-IαI in mouseserum to HA8 was also slow (≧24 hrs, panel C). Whereas IαI was thepredominant HC-donor in human serum, mouse serum contained predominantlypre-IαI with IαI as a minor component. We have observed this in multiplesubjects (not shown). The relative molecular weights of IαI and pre-IαIfor human HC1 (A) and HC2 (B) show that HC2 is about 10 kDa larger thanHC1. The IαI and pre-IαI bands from HC2 are about 20 kDa and 10 kDalarger than the IαI and pre-IαI bands of HC1. This implies that the IαImay be a homodimer (HC1-HC1 or HC2-HC2) instead of a heterodimer(HC1-HC2). Since HC transfer of HC1 is faster than HC2, HC2 transferfrom pre-IαI is not simply the result of slower transfer from pre-IαIthan IαI, but is HC specific. HC1 staining was completely absent inmouse serum (not shown). HC3 was found in mouse, but not human serum,albeit possibly because the antibody did not recognize human antigen(not shown). The effects that differential kinetics and thespecies-predominance of HC isotypes have on the development of RA, andother inflammatory diseases, is unknown.

The presence of HC5 in IαI has not been reported, but we have no reasonto believe it is not part of IαI.

It has been suggested that IαI is a heterodimer of two different HCs(HC1-HC2) (27). We have reason to believe that homodimers (HC1-HC1 orHC2-HC2) predominate, since this is the most natural interpretation ofthe 20 and 10 kDa gel shifts between IαI and pre-IαI (respectively)(FIG. 18). While we cannot conclude from the relative staining of HC1and HC2 that HC1 predominates in humans while HC2 predominates in mice,it at least raises this possibility. This is an important distinctionbecause our data show that the transfer of HC1 by TSG-6 is significantlyfaster than the transfer of HC2 to HA (FIG. 18). We also found that IαIpredominates in human blood while pre-IαI (lacking one of the two HCs)predominates in mouse blood (FIG. 18). Whether this species-specificdifference in the HC composition of IαI has an effect on the onset andprogression of RA is unknown.

Example 5 Intra-Articular Injections of HA16K and HA50K in RabbitsSubjected to Mono-Articular RA

We hypothesize that injection of HA16K (an irreversible HC acceptorhaving a molecular weight of approximately 16 kDa) into the inflamedjoint will result in the covalent transfer of HCs from pathologicalHC-HA to HA16K via endogenous TSG-6. We also hypothesize that removal ofHCs from HC-HA in the matrix of the joints will cripple this novelextracellular-matrix-based leukocyte retention mechanism, therebydecreasing inflammation in the joint.

A method has been developed to test this hypothesis. In accordance withthis method, intra-articular injections of HA16K (only a HC acceptor,having a molecular weight of 16 kDa) and HA50K (both a HC acceptor anddonor, having a molecular weight of 30.6 kDa as determined by MALLS-SEC)into rabbits subjected to mono-articular RA using the methylated BSAmodel of antigen-induced-arthritis (AIA) are carried out. The effectthat HA16K has on relieving joint swelling and inflammation compared toHA50K which functions as a negative control is compared. The method isas follows.

Manufacture Gram-Scale Quantities of Hyaluronan (HA) That are BothReversible (HA50K) and Irreversible (HA16K) Heavy Chain (HC) Acceptors.

HA16K and HA50K polysaccharides are produced in 2-gram quantities bychemoenzymatic synthesis (Hyalose LLC, Oklahoma City, Okla.). Methods,both published (20) and patented (U.S. Pat. No. 7,223,571, which isincorporated herein by reference), based on extension of short HA chains(or primers) into much longer HA chains by recombinant Escherichiacoli-derived HA synthase of Pasteurella multocida (PmHAS), will be used.Unlike other HA synthases, PmHAS can elongate exogeneously-suppliedshort HA chains (e.g. 4 sugars) into longer HA chains (e.g. 5 to 10,000sugars) (6). The HA oligosaccharide primer synchronizes thenon-processive PmHAS polymerization reaction; by synthesizing all thechains in parallel, the final sizes are all very similar or essentiallymonodisperse (e.g. polydispersity of less than 1.10). To control thesize of any polymer preparation, the stoichiometry of the primer to theUDP precursor is adjusted (e.g., more primer will yield many shortchains, and conversely, less primer will yield fewer long chains). Thismethod has been performed at the ˜0.1-gram scale for the SelectHA™product line (see http://www.hyalose.com/technology/selectha.html).Larger reaction vessels, solid phase extraction (rather than solvents),and tangential flow filtration (instead of dialysis) are employed in thescale-up process to obtain 2-gram batches. The polymer size is monitoredby multi-angle light scattering and agarose gel analyses (20). The HApurity is analyzed by the carbazole assay for HA, Bradford assay(Pierce) for residual protein, and the Limulus amebocyte assay (Cambrex)for potential endotoxin. Purity specifications accepted for FDA-approvedhigh molecular weight HA are used in viscoelastic supplementation of theknee as a benchmark. The 2-gram batch size is expected to provide moreHA than is needed for the rabbit study (˜340 mg each polymer) to allowmore thorough analytical testing and to reduce the perceived risk in themanufacturing process.

Determine the Feasibility of Treatment of RA in a Large-Animal Model byIntra-Articular Injection of HA Polysaccharides of a Size Known toFunction as an Irreversible HC Acceptor.

A rabbit efficacy study is performed, for example, by a clinicalresearch organization that provides expert regulatory, clinical, andcompliance services to medical device and healthcare productmanufacturers. A mono-articular model of RA is used. This model inducesarthritis in single SJs by locally injecting methylated bovine serumalbumin (mBSA) into knee joints of a rabbits that have been previouslysensitized to this antigen. This method has been previously described(21) and is known as antigen-induced arthritis (AIA). This method is notrabbit strain dependent, and the percent incidence of arthritisinduction is 100%. This mono-articular model has been chosen, as opposedto a systemic RA model, because it is reasonable to expect that theamount of HA polysaccharide required to have an effect on HC-HA in amono-articular model will be much less than the amount of polysaccharideneeded in a systemic model.

Immunization to mBSA:

Rabbits are immunized with 0.5 ml of mBSA homogenized 1:1 with Freund'scomplete adjuvant at 4 mg/ml. Injections are given subcutaneously at twodifferent sites. Rabbits are subcutaneously boosted with 0.5 ml of themBSA adjuvant twice weekly for 3 weeks. Two to three weeks after thefinal boost, serum anti-BSA titers are determined by ELISAs.

Induction of Arthritis:

Knees of mBSA-immunized rabbits are injected intra-articularly underketamine/xylazine anesthesia. Rabbits receive 500 μl intra-articularinjections of mBSA in saline at 2 mg/ml once weekly for 3 weeks.Typically, rabbits develop swelling and redness in mBSA treated joints 7to 14 days after the third injection of mBSA. This stage of immunizedanimals (from 7 days after the third injection) is regarded as thepre-arthritic phase of AIA. A standard scoring system, based on swellingand redness of paws, is used for the assessment of the severity ofarthritis. The time of appearance of swelling and redness are recordedas the time of onset of arthritis. Joint swelling is scored from 0 to 4of each knee. During the treatment period, knee (joint) thicknesses atfrontal and sagittal planes is measured daily with a micro-caliper (26).Two weeks after the final injection of mBSA, body weights are recordedand the knee joints injected with 500 μl of treatments (HA16K or HA50K)or controls (saline). These injections are repeated once per week for 3weeks (total of 3 injections per knee; similar to current viscoelasticHA injection protocols).

HA Polysaccharide Treatments:

Typically, about 1 ml of SF accumulates into the inflamed rabbit kneejoint with HA concentrations at about 4 mg/ml. In our in vitro studies,adding equivalent amounts of HA polysaccharide that is an irreversibleHC acceptor (such as HA16K) to the same amount of HMW HA that issubstituted with HCs (i.e. HC-HA) is sufficient to remove >90% HCs fromHC-HA. However, in vivo, we expect that LMW HA such as HA16K willdiffuse out of the synovial space into the lymphatics, thus possiblyrequiring larger dosages. Since HA is highly soluble and HA solutionsthat are 20 mg/ml can be prepared, rabbits are injected with 0.5 mlHA16K or HA50K at 20 mg/ml, once weekly for 3 weeks. This would beexpected to provide about 2.5×HA equivalents for HA polysaccharide withrespect to the amount of endogenous HA. Thus, approximately 360 mg ofHA16K and HA50K is needed to inject 12 knees 3× with 10 mgs/knee.

Harvest of Samples and Data Analysis:

Following the 3 weeks of treatment, blood, SF and tissue are harvested.Knees from all rabbits are fixed in 10% formalin containing 5%cetylpiridinium chloride (to prevent solubilization of HA), decalcifiedin 5% formic acid, paraffin embedded, and sections processed for routinehistology and immunohistochemistry. SF is collected from the kneesynovial spaces of sacrificed rabbits using a syringe. Since the amountof SF in the non-inflamed joints of untreated rabbits is small 100 μl),a 400 μl saline lavage is used. Blood is collected from sacrificedrabbits by cardiac puncture. Leukocyte populations are counted on anAdvia 120 Hematology analyzer from blood collected by cardiac puncture.Leukocyte populations from pooled SF are counted on a hemocytometer anddifferent leukocyte populations are evaluated by standard histologyusing Wright-Giemsa staining. The amount of HC-HA present in SF isevaluated by monitoring a molecular weight gel shift of theHCs+/−Streptomyces hyaluronidase treatment in a Western blot assay. Theregional distribution of HC-HA within the synovial tissue itself is alsoevaluated by immunohistological co-localization of HC antibodies withthe biotinylated HA binding protein.

Example 6 Removal of HCs from Pathological HC-HA Matrices by HA Oligos

Methods have been developed to investigate the parameters by which HAoligos remove HCs from pathological HC-HA matrices to disrupt HA-basedleukocyte binding and activation.

Synthesis of Artificial HC-HA:

A method for making artificial HC-HA has been developed usingrecombinant TSG-6 (rTSG-6) to transfer HCs from purified IαI and acommercial source of HA that is 1000 kDa. This Select HA™ (Hyalose,Inc.) is essentially monodisperse (e.g. polydispersity of 1.007), suchthat each HA strand is 1000 kDa, contrasted with standard commercialpreparations that are highly polydisperse, where HA strands can rangefrom 500 to 1500 kDa. This Select HA is chosen to minimize variableresults that might occur between commercial preparations of polydisperseHA. IαI is purified from human serum as described previously (28).Recombinant TSG-6 is prepared as previously described (29), and is alsocommercially available in both human and mouse forms.Streptavidin-coated 96-well plates, and the biotinylated HA bindingprotein (HABP) are commercially available and are used to capture HC-HAin each well for leukocyte binding studies (17). In parallel cultures,fluorophore-assisted-carbohydrate-electrophoresis (FACE) is used toquantify the amount of HA bound to each well to prove that thevariations seen in leukocyte binding are caused by changes in thenumbers of HCs present on HA rather than variations in the amount of HApresent in each well. HCs are removed from the HC-HA in the wells ofthese plates, as described in below. The effect this has on leukocyteadhesion is monitored, as also described below.

Removal of HCs from HC-HA by HA Oligos:

HA oligos eight monosaccharides long (HA8) are used to remove HCs fromHC-HA. These oligos are commercially available with a high degree ofpurity from other oligos. HA8 is the smallest HA oligo that can acceptHCs and function as an irreversible HC acceptor. To document the amountof HCs removed by HA8 from HC-HA in the well, HCs are extracted fromeach well using Streptomyces hyaluronidase (that is specific only forHA) and the amount of HCs extracted are measured by Western blot (asshown in FIG. 20).

Specifically, an analysis of heavy chain-hyaluronan complex in mouseproteoglycan-induced arthritis synovial fluid (PGIA SF) is shown (FIG.20). Evidence is provided for HC transfer onto high molecular weight(HMW) HA in mouse PGIA SF. Both knee (lanes 1-2) and ankle (lanes 3-4)mouse PGIA SF were digested for 24 hours at 37° C. with (+) or without(−) hyaluronidase (Hyase), and analyzed by Western blot analysis probingwith a mixture of anti-mouse HC1 and HC2 antibodies. Withouthyaluronidase treatment (lanes 1,3), only a pre-IαI band is seen as theHCs, attached to the HMW SF HA (HMW HC-HA) are too large to enter thegel. In lanes 2 and 4, hyaluronidase-released HCs are easily detected inPGIA mouse SF similar to that seen in human RA SF and AIA mouse SF (notshown).

It is expected that HCs below the surface of the HC-HA matrix, andespecially the HC-HA that is directly in contact with HABP at the wellinterface, are unlikely to be removed by the HA oligos, so it would notbe expected that that all HCs could be removed from these cultures.Rather, the aim is to use HA oligos to remove enough HCs from HC-HA suchthat leukocyte adhesion to this HA matrix is significantly diminished.It is expected that prevention of ≧90% of leukocytes from binding toHC-HA could be accomplished. This percentage may be less when HA8 isapplied to leukocyte cultures already bound to HC-HA since it is not yetknown if HA oligos would be as efficient at displacing bound leukocytescompared to preventing their initial binding to HC-HA.

Synthesis of HC-HA Derived from Synoviocytes:

Human synoviocytes from normal and RA patients are acquired from acommercial source (Cell Applications, Inc.). While it is possible thatthese cells would spontaneously produce leukocyte-adhesive HAstructures, such a phenomenon is uncommon (30) while all other reportedcell types require stimulation with a viral mimic, toxins or cytokinesto induce the synthesis of leukocyte-adhesive HA structures. Thus, it isexpected that cultured synoviocytes would require stimulation to produceHC-HA. Cultured synoviocytes have been shown to produce TSG-6 followingTNFα stimulation, which may induce the formation of HC-HA (31). Otherstimulants that may induce HC-HA formation are RA-related cytokines,such as IL-1, IL-6, IL-15 and IL-17. The ability of culturedsynoviocytes to produce HC-HA in response to these cytokines is tested.If these cytokines fail to induce HC-HA synthesis, the viral mimicpoly(I:C) is used. poly(I:C) is well known to induce the formation ofleukocyte-adhesive HA structures in cultured cells (2, 32). The relativeamount of HCs in the HC-HA structures produced by synoviocytes isconfirmed by Western blots of Streptomyces hyaluronidase extracts fromthese cells (as shown in FIG. 20). The effect that addition of rTSG-6 tothe culturing media has on promoting the formation of HC-HA by thesecells is also tested (as shown in FIG. 19). This procedure is used to:(i) induce synoviocytes to produce HC-HA, (ii) use HA8 to remove the HCsfrom HC-HA produced by these cells, and (iii) monitor the effect thatremoval of these HCs from HC-HA has on leukocyte binding. Using similarconcentrations and durations of HA8 as learned above, HA8 is used toremove HCs from HC-HA produced by cultured synoviocytes, similar to theprocedure described above. This is done both before and after leukocyteadhesion to determine if HA8 can both prevent and disrupt leukocytebinding to HC-HA, respectively. The procedure for leukocyte adhesion isdescribed below.

HC-HA Derived from Human and Mouse SF:

We have shown that SF derived from human patients and mice with RAcontain pathological HC-HA (FIG. 20) and that HA oligos can be used toremove almost all of the HCs from this matrix in vitro (FIG. 14). HC-HAderived from the SF of RA patients significantly promoted leukocyteadhesion compared to HA alone (17). This suggests that removal of HCsfrom HC-HA by irreversible transfer to HA oligos should have a similareffect at reducing leukocyte adhesion to SF-derived HA. Accordingly, inthis method HA8 is used remove HCs from SF-derived HC-HA, before andafter leukocyte adhesion, to evaluate the effect it has to prevent anddisrupt leukocyte adhesion to this pathological matrix. This assay isperformed essentially as described previously (17), where HC-HA from theSF of human patients and mice with RA is captured on HABP-coated plates,identical to the procedure described for artificially created HC-HAabove. HCs are removed from the HC-HA in the wells of these plates, asdescribed above. The effect this has on leukocyte adhesion is monitoredas described below.

Leukocyte Adhesion Assay:

The monocytic U937 cell line is used for initial leukocyte bindingstudies since this cell line readily binds HC-HA (FIG. 19) (33) anddemonstrates CD44 capping, indicative of activation, when bound to HAcables. These cells are fluorescently labeled with Dil (a lipophilicmembrane stain) and the number of U937 cells bound to HC-HA isdetermined by comparing the relative intensity of Streptomyceshyaluronidase extracted cells to a known number of fluorescently labeledU937 cells on a fluorometer (FIG. 19). From previous studies (notshown), we know that 2 million U937 cells per/ml, adding 1 ml for every4 cm² of surface, area is ideal. This concentration provides an adequatemonolayer of U937 cells to cover the entire surface area of the well.

Adhesion of different Leukocyte Populations to HC-HA:

Nothing is known regarding the ability of HC-HA to promote theaccumulation of distinct leukocyte populations in the inflamed joint.Our hypothesis is that not all leukocyte populations bind HC-HA withequal avidity. A method to investigate this hypothesis, by monitoringthe effect disruption of HC-HA has on the accumulation of differentleukocyte populations in SF and the inflamed joint tissue, is asfollows. Mixed leukocyte populations, derived from whole blood, areapplied to artificial HC-HA, HC-HA produced by synoviocytes, and HC-HAderived from human patients and mice with RA. Flow-cytometry is used tomeasure the relative percentages of leukocytes that bind to HC-HA,comparing these percentages to the percentages of leukocytes originallyapplied to HC-HA. Mixed leukocyte populations are purified from wholemouse and human blood using the “buffy coat” procedure of densitygradient centrifugation. The relative percentage of leukocytes in humanblood is 4-6% monocytes, 50-70% neutrophils, 25-35% lymphocytes, 1-3%eosinophils, and 0.4-1% basophils. The mixed population of leukocytes isallowed to bind HC-HA by allowing them to settle, by gravity, onto HC-HAunder static conditions (30 min). Unbound leukocytes are removed fromHC-HA bound cells by washing and are saved in a separate tube as theunbound fraction. Bound cells are released from HC-HA by a 5 mintreatment with Streptomyces hyaluronidase and saved in a separate tubeas the bound fraction. To determine the relative percentages ofleukocyte populations that preferentially bind HC-HA, (i) an aliquot ofthe leukocytes originally applied to HC-HA is compared to (ii) theunbound fraction, and (iii) the bound fraction.

Leukocyte Activation by HC-HA: We showed that monocytic leukocytes (U937cells) demonstrate signs of activation when bound to leukocyte-adhesiveHA structures produced by gut smooth muscle cells, including “capping”of the HA receptor CD44 on their cell surface and subsequent digestionof the pathological HA matrix. Nothing is known regarding the effectthat leukocyte engagement of HC-HA has on the induction ofpro-inflammatory cytokines and chemokines. Our hypothesis is thatleukocytes bound to HC-HA are stimulated to produce an array ofinflammatory mediators that regulate inflammation in the joint. A methodto evaluate the gene expression and protein levels of several cytokinesand chemokines produced by mixed populations of leukocytes bound toartificial HC-HA, HC-HA produced from cultured synoviocytes, and HC-HAfrom human and mouse SF, is as follows. Mixed populations of leukocytesfrom human and mouse blood are isolated and allowed to bind HC-HA,washing away unbound leukocytes. The leukocytes are incubated at 37° C.in the presence of HC-HA for 2, 4, 6, and 12 hours. A portion ofleukocytes not applied to HC-HA, and the portion of leukocytes that areapplied to HC-HA but do not bind to it, are also incubated at the sametemperature and time points to serve as negative controls. Positivecontrols include input, bound, and unbound fractions treated withlipopolysaccharide. Cytokine and chemokine gene expression are evaluatedby quantitative real-time PCR and protein levels are monitored byflow-cytometry using commercially available kits (BD Biosciences),including the cytokines IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70 and thechemokines IL-8, RANTES, IFNγ, □MCP-1 and IP10. If mixed populations ofleukocytes demonstrate an induction in these inflammatory mediators, HAoligos are used to remove HCs from the HC-HA matrices (as describedabove) and the effect this has on preventing this induction isevaluated. Furthermore, if mixed populations of leukocytes show changesin cytokine and/or chemokine production, flow-cytometry is used toisolate individual leukocyte populations, they are allowed to bindHC-HA, and the effect this interaction has on the cytokine and chemokineproduction by these individual leukocyte populations is examined.

Example 7 Evaluating the Effect of Disruption of Abnormal Matrix byAdministration of HA Oligos

A method for evaluating the effect of disruption of abnormal matrix byadministration of HA oligos is as follows. Normal mice are subjected tomono-articular RA to allow HC-HA to form in the SF of inflamed joints.Subsequently, the ability of HA8 to remove HCs from HC-HA matrices in SFis monitored and the effect this has on the accumulation and activationof leukocytes is evaluated. HA8 is labeled with Alexa Fluor 594 (red)and 488 (green) hydrazine (Molecular Probes, Inc.) for ease of trackingand quantification. These fluorophores attach to the reducing terminusof the oligo, such that there is one fluorophore per HA oligo to permitquantitative and linear measurements of the oligo. The labeled oligosare purified using Sephadex G-50 and their functionality as anirreversible HC-acceptor is tested with non-labeled HA8 as a positivecontrol. In the unlikely event that the fluorophores compromise theability of the HA8 to function as an irreversible HC acceptor, asuitable replacement fluorophore would be identified and/or a slightlylonger HA oligo than HA8 that might circumvent this adverse side effectwould be chosen. The route of administration for recombinant F-HA8 isintravenous, using (i) a catheter in the jugular vein, preinstalled byJackson Laboratories, and/or (ii) an osmotic pump, pre-implanted byJackson Laboratories. It is expected that the modestly higher cost ofthese pre-installed catheters and pumps will be offset by thereproducibility of the repeated injections, and blood draws, that willbe required for such a study. A single 100 μl intravenous injection ofdoses ranging from 1-10 mg/ml is made. The intravenous osmotic pump isalso tested at similar concentrations. Circulating levels of F-HA8 aremeasured by drawing 25-50 μl of blood from the intravenous catheter 2,4, 6 and 12 hrs after the initial injection, followed by daily draws, ifnecessary, until F-HA8 levels drop below the level of detection. Urineis collected by picking up the mice by the skin of their neck andletting them urinate onto a piece of Parafilm™, after which the urine istransferred to a tube using a pipet. Oligo levels are quantified bymeasuring an aliquot of serum and urine in a fluorometer, comparing thesignal to known standards of F-HA8. It is expected that the best dose,and route of administration, to reach oligo blood levels of 0.1 mg/mlover a period of 2-4 days can thereby be determined. These metrics arebased on calculations from in vitro dose-response and kinetic studies ofHC swapping from HC-HA onto HA oligos (not shown). Although the additionof recombinant TSG-6 accelerated the removal of HCs from HC-HA,endogenous levels of TSG-6 were sufficient to remove about half of theHCs in 72 hrs (FIG. 14). Thus, it is expected that endogenous TSG-6should be sufficient to deplete HC-HA within 2-4 days. The effect thatintravenous injections of recombinant TSG-6 have in accelerating thisprocess is also tested (similar to the intravenous injections of TSG-6described in (29)). Once the optimum route of delivery and dosing toachieve these circulating HA8 levels are determined, Balb/C mice aresubjected to a mono-articular model of RA. The oligos are injectedbefore and after the onset of inflammation, and the effects that HA8 hason levels, activity, and distribution of TSG-6, HA, and HC-HA and oncounts and populations of leukocytes are determined. Specifically, theoligos are injected (i) at the same time as the intra-articularinjections of mBSA (before the onset of inflammation), or (ii) sevendays after the intra-articular injection of mBSA (after the onset ofinflammation). The effect that these two approaches have on theformation or disruption of HC-HA and the subsequent effect this has onthe accumulation and activation of leukocytes in the inflamed joint aredocumented.

Example 8

Another example embodiment relates to the controlled and extendedrelease of the LMW HA oligosaccharides in tissues through encapsulationin tyramine-based (TB) HA hydrogels.

We have determined that tyramine-substituted (TS) HA can be modifiedwith HC in vitro via recombinant TSG-6 and serum as a source of IαIsimilar to “naked” HA (FIG. 9). Specifically, results from Western blotsdemonstrate that HCs from IαI were transferred to a commercial source ofHA (panels A,C) and T-HA (panels B,D) via enzymatic transfer by TSG-6.By systematically (1:4) decreasing the amount of HA in the reactionmixture while keeping the amount of the HC donor, IαI, in serum andTSG-6 concentration constant, we have created HC-HA complexes which varyin HC density on the HA backbone. The HA-bound HC have such a largecombined molecular weight (>1700 kDa) that they are unable to enter thegel (panels C,D). Hyaluronidase treatment (panels A,B) releases the ˜75kDa HC from the large HA molecule, permitting them to run as a distinctband on an SDS-PAGE gel.

The resulting HC-TS-HA can be subsequently cross-linked with horseradishperoxidase and hydrogen peroxide to form HC-TB-HA hydrogels (data notshown). These HC-TB-HA hydrogels can be used for various tissueengineering and repair applications with them engineered to bepatient-specific through the use of a patient's own serum as the sourceof IαI.

With respect to the new combinatorial materials described herein,HC-TS-HA, which refers to hyaluronan (HA) chains substituted with bothheavy chains (HC) from IαI and tyramine, and HC-TB-HA hydrogels, whichrefers to hydrogels formed from HC-TS-HA chains as a result ofdityramine formation, it appears that the transfer of heavy chains fromIαI by TSG-6 to already cross-linked TB-HA hydrogels occurs only at thesurface of the hydrogel mass. Cross-linking doesn't occur within thehydrogel mass as both the TSG-6 and/or IαI are too large to enter thehydrogel pores.

By virtue of the spacing of tyramine adducts on TS-HA chains, e.g. onetyramine every 20 repeat disaccharides (or HA40), the size of theintervening stretches of HA oligosaccharide between tyramine adducts(HA39) may function similar to LMW HA, which serve as acceptors for HCswapping, but not as donors (i.e. irreversible acceptors of HC).Currently, HA molecules ranging from 8-14 monosaccharides in length havebeen found functional in the disclosed methods, although it is believedthat HA molecules of greater length, e.g. to less than about 160monosaccharides, more preferably to 40 monosaccharides would also workto serve as LMW HA to achieve irreversible HC attachment via TSG-6.Regardless of the upper bound for LMW HA useful in the disclosedmethods, TS-HA, unlike native or naked HA, may be a form of highmolecular weight (HMW) HA, that is an irreversible acceptor of HCsimilar to the low molecular weight (LMW) HA oligosaccharides that arethe focus of this disclosure.

Similar to the point above, the spacing of the tyramine adducts on TS-HAchains, e.g. one tyramine every 20 repeat disaccharides (or HA40),generates upon hyaluronidase digestion oligosaccharides that arepredicted to be in the size range of those LMW HA oligosaccharides thatserve as acceptors for HC swapping, but not as donors (i.e. irreversibleacceptors of HC) similar to the LMW HA oligosaccharides that are thefocus of this invention.

Based on the work by Kim et al (34) it appears that uncross-linked TS-HAalone when injected into an RA joint in an RA animal model is able tosuccessfully treat the RA. The authors postulate that the therapeuticeffect is derived from the ability of the TS-HA chains to scavengeoxygen free radicals known to be present in RA joints and previouslyshown to cross-link TS-HA chains into TB-HA hydrogels in the absence ofHRP or hydrogen peroxide. However, in light of the observationsdescribed above, it is expected instead that the therapeutic effect ofthe TS-HA chains is that the TS-HA, either as HMW TS-HA chains or uponfragmentation by free radicals or the action of hyaluronidase to HAoligosaccharides, serve as a source of irreversible HC acceptors toremove HC from endogenous synovial fluid HA and thus remove the bindingand activation signal for inflammatory cells that perpetuates thedisease.

One embodiment for treatment (whether injectable, inhalable, digestible,topical, etc.) of an inflammatory condition is encapsulation of nakedLMW HA oligosaccharides (that can serves as acceptors for HC swapping,but not as donors) within TB-HA hydrogels designed to extend the releaseof the naked HA oligosaccharides over a 30 day or other desirable periodsimilar to the previously reported in vivo release of dexamethasone.This therapeutic release of naked HA oligosaccharides would beresponsible for the initial removal of HCs from the synovial fluid HMWHA and relief from symptoms (and possibly disease). Then the slowdegradation of the TB-HA hydrogel would provide for release of TS-HAoligosaccharides as irreversible HC acceptors over a longer time periodso as to extend the relief from symptoms (and possibly disease) perhapsout 3-12 months. In this manner, a novel therapy would be available toprovide a treatment that provides relief from chronic inflammation thatdoes not act by directly altering immune cell function and thus produceassociated complications, and is either simple to administer frequently(i.e. through a topical lotion, ingestion of a pill, an inhaler) oradminister occasionally only every 3, 6 or 12 months (i.e. transfusion,intra-articular injection) as a minimally invasive, outpatientprocedure.

Example 9

Another example embodiment is intra-articular injection of LMW HAencapsulated in TB-HA hydrogel into the joints of rheumatoid arthritispatients. The hydrogel can be engineered to release the LMW HA over aspecified time period (e.g. 1 month) at therapeutic levels required totransfer endogenous HC from synovial fluid HA to the LMW HA. This isaccomplished through endogenous TSG-6 already present in the synovialfluid. The LMW HC-HA is subsequently cleared rapidly from the jointspace removing the signal responsible for binding and activation ofinflammatory cells thus reducing the signs and symptoms of the disease,and ultimately helping slow or halt progression of further tissue damagein the joint. Our experiments (FIG. 9) show that TSG-6 does transfer HCto (TS) HA. However, it does not transfer HC once the (TS) HA is firstcross-linked to form a hydrogel (data not shown). Thus the hydrogel actsas an inert carrier until it is eventually degraded. Its degradation ispredicted to result in formation of HA and (TS) HA oligosaccharides withsufficiently small size to be unable to serve as a donor for HC transferthus augmenting the encapsulated LMW HA.

The foregoing experiments have confirmed: 1) the irreversible nature ofthe transfer of HC from HMW HA to LMW HA using recombinant TSG-6; 2) theability to transfer HC ligands from HA of synovial fluid of rheumatoidarthritis patients to LMW HA using recombinant TSG-6; and 3) the abilityto transfer HC from serum IαI to TS-HA using recombinant TSG-6 andsubsequently form HC-TB-HA hydrogels via cross-linking with peroxidaseand hydrogen peroxide.

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The invention has been described with reference to the exampleembodiments described above. Modifications and alterations will occur toothers upon a reading and understanding of this specification. Examplesembodiments incorporating one or more aspects of the invention areintended to include all such modifications and alterations insofar asthey come within the scope of the appended claims.

What is claimed is:
 1. A method of transferring Inter-α-Inhibitor (IαI)heavy chains (HCs) from high molecular weight (HMW) hyaluronic acid (HA)to low molecular weight (LMW) hyaluronic acid (HA), within a site ofinflammation caused by asthma in an individual in need thereof,comprising administering to the site: a) a LMW HA compound comprisingthe hyaluronan-containing structure of:

wherein: A is hydrogen; B is hydroxyl; x=0 or 1; y=0 or 1; and n=3 to70, with the proviso that if n=3, then at least one of x and y=1 and b)a protein selected from the group consisting of mammalian TSG-6 protein,recombinant mammalian TSG-6 protein, human TSG-6 protein, andrecombinant human TSG-6 protein, in amounts of a) and b) effective totransfer the IαI HCs from the HMW HA to the LMW HA compound.
 2. Themethod of claim 1, wherein the individual is a mammal selected from thegroup consisting of a human, a dog, a cat, and a horse.
 3. The method ofclaim 1, wherein n=3 to
 45. 4. The method of claim 1, wherein x, n, andy are selected from the group consisting of: (i) x=0, n=4, and y=0; (ii)x=0, n=5, and y=0, (iii) x=0, n=6, and y=0; (iv) x=0, n=7, and y=0; (v)x=0, n=9, and y=0; (vi) x=1, n=10, and y=0; and (vii) x=0, n=10, andy=1.
 5. The method of claim 1, wherein x, n, and y are selected from thegroup consisting of: (i) x=0, n=43, and y=0; (ii) x=1, n=43, and y=0,(iii) x=0, n=43, and y=1; (iv) x=1, n=43, and y=1; and (v) x=0, n=44,and y=0.